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places of Bengaluru. Statistical software: The data was entered using statistical software namely SPSS Version 25.0, Microsoft Excel 2016 and Microsoft word 2016 was used to draw tables and graphs. Discussion A cross sectional study was conducted to measure the prevalence of work-related musculoskeletal disorders and to assess the prevalence of severity of disability due to low back pain among self-employed female tailors.The study group included subjects aged above 18 years.The prevalence of low back pain was measured using Modified Oswestery Scale and the prevalence and consequences of musculoskeletal system was measured using Standardized Nordic questionnaire. A total of 70 subjects participated in this study after signing the informed consent forms.They were assessed for the variables along with the demographic data and the findings were recorded. Several cross-sectional studies stated that the low back and the musculoskeletal system were the most commonly affected among the garment workers, were at higher risk of developing musculoskeletal disorders and MSDs were highly prevalent especially in the upper extremity among sewing profession population. The findings of this study confirm the workrelated stress on musculoskeletal system on selfemployed female tailors.In the present study, it was observed that the prevalence of neck trouble in last 12 months was 47.1%, trouble during last 7 days was 50 % and ADL affected in last 12 months was 55.7%.The prevalence of Shoulder trouble during last 12 months in right shoulder was 45.7%, in left shoulder was 45.7% and both the shoulders was 1.4%.Trouble during last 7 days in right shoulder was 61.4%, left shoulder 37.1%

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and both the shoulders was 1.4%.54.3% tailors reported ADL getting affected in last 12 months. The prevalence of troubles in last 12 months in right Elbow was 55.7%, in left Elbow was 42.9% and both the Elbow was 1.4%.Trouble during last 7 days in right Elbow was 80.0%, left Elbow was 20.0% and both the Elbow was 75.7%.The ADL affected in last 12 months was 24.3 %.The prevalence of trouble in last 12 months in right Wrist was 54.3%, left Wrist 41.4% and both the Wrist was 4.3%.Trouble during last 7 days in right Wrist was 57.1%, left Wrist was 41.4% and both the wrist was 1.4%.64.3% of tailors reported that their ADL was affected during the last 12 months due to trouble in wrist.Prevalence of Upper back trouble in last 12 months was 58.6%, trouble during last 7 days was 48.6% and ADL affected in last 12 months was 71.4%.The prevalence of Low back trouble in last 12 months was 21.4%, trouble during last 7 days was 42.9% and ADL affected in last 12 months was 42.9%.The prevalence of Hip trouble in last 12 months was 50.0%, trouble during last 7 days was 50.0% and ADL affected in last 12 months was 44.3%.The prevalence of Knee trouble in last 12 months was 44.3%, trouble during last 7 days was 44.3% and ADL affected in last 12 months was 40.0%.The prevalence of Ankle trouble in last 12 months was 65.7%, trouble during last 7 days was 67.1% and ADL affected in last 12 months was 68.6%.The

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secondary objective was the find out the prevalence of low back pain using Modified Oswestery lower back disability index.The analysis of this study presented a mean score of 32.59. Scope of the Present Study Was: A high prevalence of musculoskeletal disorders exists among self-employed female tailors that affects their ADLs, productivity, and quality of work.Further studies are needed to identify the specific risk factors for the ergonomic changes to bring about and to assist in planning management strategy including awareness, education, and treatment to prevent work-related musculoskeletal disorders. Conclusion The study found high rate of musculoskeletal disorders among self-employed female tailors.More than 79.2% of tailors suffered from musculoskeletal pain and lower back were the most prevalent site.Frequent breaks during work period and back support would reduce the musculoskeletal stress on lumbar region.Based on the observations made, the study concluded that there is ample scope for ergonomic improvement keeping in view the need for maximum comfort to the tailors to promote their health and wellbeing and enhance their productivity and quality of work. Limitation Sample size could have been more in number to give a better result.Study set up can be at rural community to get better understanding of workrelated musculoskeletal disorders and lower back pain involvement.Use of questionnaire in regional language could have given better results. Data: Self-employed female tailors Definition of study subjects: Female tailors from selected places of Bengaluru based on inclusion and exclusion criteria.Inclusion Criteria: • Age of respondents>18 years • More than 8 hours of work • Subjects willing to participate and

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ready to sign consent form.Exclusion Criteria: • Subjects with neurological dysfunction, musculoskeletal dysfunction, psychiatric disorder • Subjects with gynecological conditions • Subjects who had already participated in similar kind of study.• Pregnant women and lactating mother • People with disabilities Method of Collection of Data Sampling Method: Purposive sampling technique Sampling Size: Subjects matching up inclusion and exclusion criteria.Duration of the Study:Data was collected over a period of in 3 months' time.ProcedureInvestigator personally contacted the selfemployed female tailors and subjects that fulfilled the inclusion and exclusion criteria and were recruited for the study.Interviewer presented a structured questionnaire along with a pretested checklist for assessment of the seeing workstation that were used.The questionnaire consisted of four sections; the first part included questions on sociodemographic characteristics and background information of the respondents; second part included questions related to occupational variables like working duration, years of work, hours of working per day and days per week and whether her job is part time or full time.The third part of the questionnaire dealt with the presence and pattern of musculoskeletal disorder which was assessed by Nordic Musculoskeletal questionnaire.The fourth part was to assess the severity of lower back pain by using Modified Oswestry Lower back disability Index.

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HQET quark-gluon vertex at one loop We calculate the HQET quark-gluon vertex at one loop, for arbitrary external momenta, in an arbitrary covariant gauge and space-time dimension. Relevant results and algorithms for the three-point HQET integrals are presented. We also show how one can obtain the HQET limit directly from QCD results for the quark-gluon vertex. Introduction Heavy Quark Effective Theory (HQET) is an effective field theory approximating QCD for problems with a single heavy quark having mass m, when characteristic momenta of light fields are much lower than m, and there exists a 4-velocity v such that characteristic residual momenta k = p − mv of the heavy quark are also small. It has substantially improved our understanding of heavy quark physics during the last decade [1,2]. Methods of perturbative calculations in HQET are reviewed in [3]. In this paper, we calculate the quark-gluon vertex in the leading-order HQET (1/m 0 ) at one loop, for arbitrary external momenta, in an arbitrary covariant gauge, in spacetime dimension d = 4 − 2ε. This allows us to take all on-shell limits (introducing additional 1/ε divergences) directly. The general d-dimensional results can also be used for expansion around a dimension other than 4; for example, 2-dimensional HQET was considered in the literature in some detail. A one-loop calculation of the QCD quark-gluon vertex with a finite quark mass m has been recently completed [4] (where references to earlier partial results can be found). We check how the HQET result can be obtained by taking the limit m

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→ ∞ in the QCD result. Let the sum of bare one-particle-irreducible vertex diagrams in HQET ( Fig. 1) be ig 0 t a Γ µ (k, q). The "full" momenta of the incoming quark and the outgoing one are where v is the heavy-quark 4-velocity (v 2 = 1), and k, k ′ are the residual momenta. The momentum transfer is q = p ′ − p = k ′ − k. In the HQET limit, m → ∞, k ∼ k ′ ∼ q ∼ O (1). The heavy quark propagator in HQET is and the elementary quark-gluon vertex is ig 0 t a v µ . In the leading-order HQET, heavy-quark propagators and vertices do not depend on the component of the heavy-quark momenta orthogonal to v. Therefore, Γ µ (k, q) does not depend on k ⊥ = k − (k · v)v. The only vectors in the problem are v and q, and (see [3]) Γ µ (k, q) = Γ v (ω, ω ′ , q 2 )v µ + Γ q (ω, ω ′ , q 2 )q µ , where are the residual energies, and q·v = ω ′ −ω. The functions Γ v and Γ q can be reconstructed from the contractions where is the 3-momentum transfer squared in the v rest frame. At the tree level, Γ µ = v µ . One-loop corrections are shown in Fig. 2. The contribution Γ µ a of the diagram Fig. 2a is proportional to v µ ; that

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of Fig. 2b has both structures. The contraction Γ µ (k, q)q µ can be simplified using the identities shown in Fig. 3. Here a gluon line with a black triangle at the end denotes a "longitudinal gluon insertion"; when attached to a vertex, it means just the contraction with the incoming gluon momentum (note that it contains no gluon propagator!). A dot near a propagator means that its momentum is shifted by q. The colour structures are singled out as prefactors in front of the propagator differences. The circular arrow in Fig. 3b shows the order of indices in the colour structure of the three-gluon vertex if abc . Two last terms in Fig. 3b contain longitudinal gluon insertions again; for them, the identities of Fig. 3 can be recursively used. Application of these identities to the diagrams of Fig. 2 is shown in Fig. 4. Here the non-standard vertices of Fig. 5 are ig 0 t a and g 2 0 f abc t c v µ . This is, of course, just the oneloop case of the general Ward-Slavnov-Taylor identity for the quark-gluon vertex, which is discussed in [4,5] in detail. The one-loop contributions to this identity are collected in Eqs. (2.28) and (2.29) of [4]. They can be easily associated with the diagrams shown in Fig. 4. The only term which requires some explanation is the diagram involving oneloop ghost self-energy contribution (the first diagram in the second line of Fig. 4b). Its connection with the last contribution on the r.h.s. of

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Eq. (2.29) of [4] is shown in Fig. 6. This relation can be easily verified by direct calculation. Using Fig. 6, we can avoid introducing the non-standard vertex shown in Fig. 5b. For the contributions of the diagrams of Fig. 2a Here −iΣ(ω) is given by the one-loop self-energy diagram of Fig. 7: (see [9]). In what follows, we shall not explicitly write +i0 in denominators. Here ξ = 1 − a 0 , a 0 is the bare gauge-fixing parameter. We can see that the Yennie gauge [6] (see also in [7]) is of special interest, since Σ(ω) is finite at ξ = −2. Moreover, if the generalization of Yennie gauge to an arbitrary dimension is chosen as ξ = −2/(d − 3) [8], then in the Abelian case (and, in particular, at one loop), the heavy-quark self energy vanishes [9] (see [3] for a tutorial). The two-loop HQET self energy was obtained in [9]; the three-loop one can be calculated using the methods of [10]. These calculations are based on integration by parts [11]. Using the identity (7), we can obtain the result for the diagram of Fig. 2a without calculations: This result is also confirmed by direct calculation. Feynman integrals of the type of Fig. 2a, can always be calculated, for integer ν 1 , ν 2 , by applying required number of times. We note that in [12] integrals of the type (11) with different velocities v have been examined. In Appendix A, we present general results for the Feynman integrals of

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the type of Fig. 2b, for arbitrary d and powers of the denominators. In Appendix B some issues related to the m → ∞ limit of scalar integrals occurring in QCD are examined. In Appendix C, we discuss the relation between the QCD vertex at k, q ≪ m and the HQET vertex. In Appendix D, we present the HQET vertex for the heavy-quark scattering in an external gluon field in the background-field formalism. Master integral The master integral is convergent at d = 4, except for the case q 2 = 0 when it has a collinear divergence (infrared if q = 0). We shall consider the region ω < 0, ω ′ < 0, q 2 < 0, where no real intermediate states exist. Using the HQET version of Feynman parametrization (see, e.g., twice, we have where y and y ′ have the dimensionality of inverse energy. For ε = 0 (d = 4), calculating the integral in y ′ and substituting y = 1/z, we obtain where z has the dimensionality of energy. Separating the logarithm as and making the substitution z = (−q 2 )/z ′ in the second integral, we obtain the representation which is explicitly symmetric under ω ↔ ω ′ . The poles of the denominators at z = Q ± (ω − ω ′ ) are compensated by the corresponding zeros of the numerators. Finally, we obtain This expression has cuts at ω > 0, ω ′ > 0, and Q < |ω ′ − ω|, where real

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intermediate states exist. General results Here we present the one-loop HQET vertex, for arbitrary d and ξ. The contribution of the diagram of Fig. 2a was given in (10). For Fig. 2b, we obtain where Ω is defined in (18). We have also derived the first contraction using (7) (Fig. 4). The second contraction vanishes in the Feynman gauge, because the three-gluon vertex yields 0 when contracted with v in all 3 indices. We also note that there are some cancellations in the generalized Yennie gauge, ξ = −2/(d−3), as well as in the "singular" gauge ξ = −4/(d − 4) (which was discussed in [14] in connection with the three-gluon vertex). When the bare vertex Γ µ is expressed via the renormalized quantities it should become Z Γ Γ µ r , where Z Γ = 1 + Z 1 α s /(4πε) + · · · is a minimal renormalization constant, and the renormalized vertex Γ µ r is finite in the limit ε → 0. Retaining only the pole parts I(ω) → 2ω/ε, G(q 2 ) → 1/ε, V(ω, ω ′ , Q) → 0, we obtain, either from (25) or from (26), When g 0 Γ µ = gΓ µ r Z 1/2 α Z Γ is multiplied by the external leg renormalization factors Z Q Z 1/2 A , it should give a finite matrix element. Using (8), n l is the number of light flavours), we arrive at This means that the heavy-quark coupling with the gluon field in HQET

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is renormalized in the same way as the other QCD couplings. Of course, this must be the case, because otherwise renormalization would destroy gauge invariance of HQET. q parallel or orthogonal to v In the parallel case q = (ω ′ − ω)v, Q = 0. The denominators of (19) are linearly dependent. Inserting into the integrand, we obtain exactly at any d. The vertex Γ µ is, of course, proportional to v µ . Therefore, we obtain, either from (25) or from (26), It has an imaginary part, which is contained in G((ω ′ − ω) 2 ). The case when q is orthogonal to v (ω ′ = ω, q 2 = −Q 2 ) does not lead to great simplifications. The contribution (10) The case q = 0 belongs to all the categories considered above. We obtain, from each of the above results, Γ q = 0, exactly at any d. Quark(s) on the mass shell If one of the quarks is on its mass shell, say ω ′ = 0, then at d = 4. The contractions of the vertex are obtained from (25), (26) by setting I(ω ′ ) = 0 and then ω ′ = 0. When both quarks are on shell (ω = ω ′ = 0), In this case, V(0, 0, Q) does not appear. It is easy to consider the cases when q is parallel to v and ω ′ = 0, and when q 2 = 0, ω ′ = 0. Conclusion We have obtained general

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expressions (10), (25) and (26) for the one-loop HQET quarkgluon vertex. Using recurrence relations (14)-(17), we expressed the results in terms of one non-trivial integral V(ω, ω ′ , Q) (19) and some trivial integrals (13). For the integral (19) in four dimensions, we have obtained an analytic result (24) in terms of dilogarithms. In Sections 3.2-3.4 we have also studied some special limits of interest. In Appendix A we have provided some results for the integrals (12) with arbitrary indices and in arbitrary dimension. We have also discussed, in Appendix B, how the HQET limit can be obtained directly from the standard integrals occurring in the QCD calculation. Using this prescription, in Appendix C we have examined the m → ∞ limit of the general QCD result [4] for the one-loop quark-gluon function, and we have found that it is in agreement with our calculation. We have also presented the result for the background field vertex (Appendix D). Acknowledgements. We are grateful to V. A. Smirnov for useful comments on the manuscript, and to T. Mannel and P. Osland for useful discussions. A. G.'s work was supported in part by the RFBR grant 00-02-17646. A. D.'s research was supported by the Deutsche Forschungsgemeinschaft. Partial support from RFBR grant 01-02-16171 is also acknowledged. In the special case ω = ω ′ = 0, the integral (43) can be evaluated in terms of Γ functions, In particular, for ν i = 1 we get In another special case, q 2 = 0, we get a hypergeometric function,

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Using simple transformations of 2 F 1 function, it is easy to see that the result obeys the symmetry (ω, ν 1 ) ↔ (ω ′ , ν 2 ), as it should. Note that the structure of the result (46) is quite similar to that of the two-point integral with different masses and zero external momentum, see eq. (2.9) of [15]. When ν 1 = ν 2 = 1, the result (46) reduces to Let us represent the denominator of (43) in terms of double Mellin-Barnes integral, expanding with respect to ω and ω ′ (see, e.g., Eq. (3.4) of [15]). Then, the resulting momentum integral can be recognized as where t 1 and t 2 are the contour integration variables. Using (44) we can evaluate the integral (48) in terms of Γ functions. Making a linear substitution for the contour integration variables (t 1 = s + t, t 2 = s − t), we arrive at the following double Mellin-Barnes representation for the integral (12): B QCD integrals in the HQET limit Here we discuss the relation of massive integrals occurring in standard QCD calculations and HQET integrals. First, let us consider the two-point integral with one massive line and one massless line, For general values of ν 0 , ν and d, such integrals have been examined in [16,17]. When we substitute p = mv + k, the massive denominator in (50) becomes For k ≪ m, several regions of integration in l are essential. When l ∼ k, we can expand

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the heavy propagator (51) in both k/m and l/m, and it becomes the HQET propagator. Higher terms form an expansion in k/m. Let us subtract and add this expansion of the heavy propagator to the exact one. In the difference, the contribution of small l ∼ k is suppressed; typically, l ∼ m. Therefore, we can expand this integrand difference in regular series in k/m, and integrate term by term. Integrals of all terms of the HQET integrand expansion in k/m vanish in dimensional regularization, because they contain no scale. Therefore, this "hard" contribution can be obtained by expanding the exact QCD integrand (51) in k/m, and integrating term by term. It is analytical at k = 0, by construction. The leading term is proportional to m d−2ν 0 −2ν , by dimensionality, whereas the higher terms form an expansion in k/m. This separation of J(ν 0 , ν; m, 0) at k ≪ m into two contributions [17] is a particular case of a more general threshold expansion [18]. Note that k plays a role of the threshold parameter, since p 2 − m 2 ∼ mk. We can check these qualitative considerations, using an explicit expression for J(ν 0 , ν; m, 0). It can be presented in terms of 2 F 1 function of z = p 2 /m 2 (see Eq. (10) of [16]). Note that in the HQET limit z approaches 1, i.e., it is at the border of convergence of the 2 F 1 function. Transforming from the variable z

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to 1 − z, we obtain (see also Eqs. (1.12)-(1.15) of [17]) We see that the first term here is nothing but the "hard" contribution. It has a prefactor m d−2ν 0 −2ν . The prefactor of the second 2 F 1 function is up to higher powers of 1/m. This is the HQET ("ultrasoft") contribution; in the leading The prefactors contain m d−2ν 0 −2ν 1 −2ν 2 (case (i)) and m −ν 0 (case (ii)). The series (i) yields the "hard" contribution. The HQET ("ultrasoft") contribution is given by the series (ii), whose leading term is j = 0. These two contributions are related to the singular limits 1/m = 0 and k = q = 0 in a way similar to the two-point case. Picking up this leading HQET contribution, we arrive at a double Mellin-Barnes representation (in terms of the remaining contour integrals over s and t), where the kinematical variables involved yield (in the limit m → ∞) the HQET variables, After introducing the new contour variables (s + t)/2 and (s − t)/2 we see that the resulting Mellin-Barnes representation is equivalent to (49), so that Therefore, the HQET integrals can be formally obtained directly from the standard loop integrals, by picking up the formal Taylor series in 1/m which has no prefactors containing m to the power depending on d. Such prescription is similar to some other prescriptions in dimensional regularization. For instance, considering the massless limit of the integral (50) we need to represent the result in terms

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of the functions of the variable m 2 /p 2 , and then discard the contribution containing m −2ε (see Eq. (11) of [16]). We have also demonstrated that the HQET contributions are equivalent to the "ultrasoft" ones, in the language of the threshold expansion [18]. In other words, in the cases considered the exact result is given by the sum of two contributions. The first one is given by a formal Taylor expansion of the integrand in the small parameter of the threshold expansion (the "hard" contribution). The second one is nothing but the HQET series in 1/m. C QCD vertex at m → ∞ If we substitute the "hard" parts of all scalar integrals into the QCD vertex, then, in the limit k → 0, q → 0, we just get the on-shell vertex at q = 0. Corrections to this limit are regular expansion terms in k/m, q/m. Since we do not consider 1/m suppressed terms here, these corrections can be omitted. If we substitute the HQET parts of all scalar integrals (see Appendix B), we should obtain the HQET vertex, which was calculated in Sect. 3. In order to make a strong check of both the results of [4] (where the one-loop quark-gluon vertex was calculated in arbitrary gauge and dimension) and of the present ones, we consider here the HQET limit of the QCD vertex [4]. Using the standard decomposition of the quark-gluon vertex [21] (see also in [4,22]), it can be split into longitudinal and transverse parts, Γ µ =

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Using the results for λ i and τ i listed in [4], we have obtained that at the order 1/m 0 the coefficient of the chromomagnetic structure T µ 5 vanishes, whereas those of L µ 1 and T µ 3 reproduce the results (10), (25) and (26), for arbitrary d and ξ. Thus the QCD vertex at small k, q is equal to its on-shell value at k = q = 0 plus the HQET vertex, up to 1/m corrections. We can reformulate this statement: the QCD vertex in the on-shell renormalization scheme is equal to the HQET vertex in the on-shell renormalization scheme, up to 1/m corrections. In QCD, the on-shell renormalization subtracts from the one-loop correction its value at k = q = 0. In HQET, the on-shell renormalized vertex equals the bare one, because its value at k = q = 0 vanishes.

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In Situ Experiments in the Scanning Electron Microscope Chamber Since the first scanning electron microscope by Knoll (1935) and theoretical developments by von Ardenne (1938a, b) in the 30’s, this imaging technique has been widely used by generations of searchers from all the scientific domains to characterize the inner structure of matter. Even if the obtained information is essential for matter description or comprehension of matter transformation, the main constraints associated with classical electron microscopy, i.e. the necessity to work under vacuum and the necessity to prepare the sample before imaging, have always limited the possibilities to “post mortem” characterisation of samples and avoided observation of biological samples. Introduction Since the first scanning electron microscope by Knoll (1935) and theoretical developments by von Ardenne (1938a, b) in the 30's, this imaging technique has been widely used by generations of searchers from all the scientific domains to characterize the inner structure of matter. Even if the obtained information is essential for matter description or comprehension of matter transformation, the main constraints associated with classical electron microscopy, i.e. the necessity to work under vacuum and the necessity to prepare the sample before imaging, have always limited the possibilities to "post mortem" characterisation of samples and avoided observation of biological samples. Electron microscopists early identified the necessity to undergo these limits. The development of a SEM chamber that is capable of maintaining a relatively high pressure and that allows imaging uncoated insulating samples began in the 70's and has been "achieved" in the late 90's -early 00's (Stokes, 2008)

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with the commercialisation of the low-vacuum and environmental SEM. The availability of new generations of electron guns (and more particularly the field effect electron gun characterized by a very intense brightness), as well as the new generation of electronic columns that are now commonly associated with the environmental scanning electron microscopes opens new possibilities for material characterisation up to the nanometer scale. The development of this generation of microscopes have opened the door for performing real time experiments, using the electron microscope chamber as a microlab allowing direct observation of reactions at the micrometer scale. Many SEM providers or researchers have developed specific stages that can be used for the in situ experimentation in the scanning electron microscope chamber. This field is one of the most interesting uses of the ESEM that offers fantastic opportunities for matter properties characterisation. Even if numerous recent articles and reviews are dedicated to in situ experimentation in the VP/ESEM (Donald, 2003 ;Mendez-Vilas et al., 2008 ;Stokes, 2008 ;Stabentheiner et al., 2010 ;Gianola et al., 2011 ;Torres & Ramirez, 2011), no one describes all the possibilities of this technique. The present chapter will provide a large -and as exhaustive as possible -overview of the possibilities offered by the new SEM and ESEM generation in terms of "in situ experiments" focussing specifically on the more recent results (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011). This chapter will be split into five parts. We will first discuss the goals of in situ experimentation. Then, specific parts will be devoted to in situ mechanical tests, experiments www.intechopen.com under wet conditions,

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and a forth part dedicated to high temperature experiments in the SEM. Last, a specific part will be devoted to the "future" of in-SEM experiments. In each part, the main limits of the technique as well as the detection modes will be reported. Each part will be focussed on examples of the use of the technique for performing in situ experiments. Goals and implementation requirements of in situ experimentation The main goal of in situ experimentation in the SEM (or ESEM) chamber is to determine properties of matter through the study of its behaviour under constraint. This requires the combination of data collection over a given duration (on a unique sample) and image treatment for information extraction. The studied properties are generally related to microscopic phenomena and hardly assessable by other techniques. In situ experiment in the SEM chamber corresponds to both imaging systems in evolution under a constraint and imaging systems stabilized under controlled conditions. To achieve this goal, several requirements are necessary: • The duration of the phenomenon to be observed must be suitable with the image recording time. If the system evolution is too fast, it will be impossible to record several images and observe this evolution. At the contrary, if the reaction kinetic is low, the time necessary for image recording will be too long and incompatible with experimentation. The high and low limits can be estimated ranging between 2 minutes and 48 hours. • The system must remain stable under the environmental conditions and/or irradiation by the electron beam during the

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time necessary for image recording. In the case of easily degradable samples, it is necessary to adjust the imaging conditions (high voltage, beam current, aperture, working distance, detector bias…) constantly, as the sample environmental conditions are modified during the experiment. Thus, the effect of the electron beam on the sample morphology modifications must be verified. Some authors report that it can act as an accelerator (Popma, 2002) or inhibitor (Courtois et al., 2011) of the observed reactions. • The image resolution must fit well with the size of details to be observed. Improvements in the image resolution have been achieved in the last decade thanks to the field effect emission guns. However, the presence of gas in the VP-SEM/ESEM chamber contributes to the incident electron beam scattering and subsequent degradation of the image resolution. Thus, the acquisition conditions must be adapted to the sample to be studied depending on the higher magnification to be reached. • The gaseous environmental conditions in which the studied system evolutes (or can be stabilized) must be reproduced in the SEM/LV-SEM/ESEM chamber. The development of the ESEM offers real new opportunities in term of composition of the atmosphere surrounding the sample. The large field detector and the gaseous secondary electron detector (Stokes, 2008) have been developed specifically for imaging under "high pressure" conditions (up to 300Pa and 3000Pa respectively) whatever the gas composition (air, water, He, He+H 2 mixtures, O 2 ). Other detectors have been developed for very specific applications (high temperature under vacuum (Nakamura et al., 2002), EBSD at

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high temperature (Fielden, 2005)). www.intechopen.com • The constraint in which the studied system evolutes (or can be stabilized) must also be reproduced in the microscope chamber. Some devices are commercialized by official sellers. Among them, we must report the Peltier stage for temperature control in the -10 to 60°C range, hot stages for temperature control up to 1500°C, stages for mechanical tests (Figure 1). Some authors have developed their own specific stages adapted to the problem to be treated (Fielden, 2005;Bogner et al., 2007). However, the development of miniaturized stages that can be positioned in the SEM chamber without creating perturbations on the incident electron beam can be really challenging. This will probably be a key in the development of in situ experimentation in the next years (Torres & Ramirez, 2011). The basis of in situ experimentation in the SEM is the study of the morphological modifications of the sample under constraint. Thus, this requires recording of numerous high quality images for image post treatment and data extraction in order to characterize the reaction or matter properties. The sample size can vary from 1µm to 50mm, and the image resolution is in the 1-10nm range, depending on recording conditions. The images are SEM images, i.e. with a large depth of field and with grey level contrasts. In-SEM experimentation can be extended to a wide range of applications, corresponding to very different materials (plants (Stabentheiner et al., 2010), food (Thiel et al., 2002 ;James, 2009), paper (Manero et al., 1998), soft matter, polymers, metals, ceramics, solids, liquids…)

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or problems (plant behaviour, chemical reactivity, properties characterization, sintering, grain growth, corrosion…). In the literature, the main part of the data reported has been acquired using an environmental scanning electron microscope. Boehlert (2011) have recently underlined the interest of performing in situ mechanical tests in the SEM and summarized it as follows. "In situ scanning electron microscopy is now being routinely performed around the world to characterize the surface deformation behavior of a wide variety of materials. The types of loading conditions include simple tension, compression, bending, and creep as well as dynamic conditions including cyclic fatigue with dwell times. These experiments can be performed at ambient and elevated temperatures and in different environments and pressures. Most modern SEMs allow for the adaptation of heating and mechanical testing assemblies to the SEM stage, which allows for tilting and rotation to optimal imaging conditions as well as energy dispersive spectroscopy X-ray capture. Perhaps some of the most useful techniques involve acquisition of electron backscatter diffraction (EBSD) Kikuchi patterns for the identification of crystallographic orientations. Such information allows for the identification of phase transformations and plastic deformation as they relate to the local and global textures and other microstructural features. Understanding the microscale deformation mechanisms is useful for modeling and simulations used to link the microscale to the mesoscale behavior. In situ mechanical tests In turn, simulations require verification through in situ microscale observations. Together simulations and in situ experimental verification studies are setting the stage for the future of material science, which undoubtedly involves accurate prediction of

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local and global mechanical properties and deformation behavior given only the processed microstructural condition". As a direct consequence of the great interest of the collected information, many different works from several scientific domains have been published for long. Thiel & Donald (1998) and Stabentheiner et al. (2010) describe the deformation of plants (carrots and leaves respectively) during room temperature tensile tests performed in the ESEM chamber. Similar tests are also reported with food (Stokes & Donald, 2000) and they are regularly performed on polymers (Poelt et al., 2010;Lin et al., 2011), composites (Schoßig et al., 2011) and metals (Boehlert et al., 2006;Gorkaya et al., 2007). Mechanical tests on metals, alloys and ceramics can also be performed at high temperature (Biallas & Maier, 2007;Chen & Boehlert, 2010). High temperature EDSB, developed by Seward et al. (2002), offers the possibility to observe phase transformations in materials as a function of temperature, as well as the direct visualization of the associated microstructural modifications (Seward et al., 2004). Several recently developed techniques allow characterizing materials at the nanometer scale through both technological miniaturization and advancements in imaging and small-scale mechanical testing. Ahmad et al. (2010) have developed a coupled ESEM-atomic force microscope to characterize single cells mechanical properties ( Figure 2). This ESEMnanomanipulation system allowed determining effects of internal influences (cell size and growth phases) and external influence (environmental conditions) on the cell strength. Gianola et al. (2011) reports the development of a quantitative in situ nanomechanical testing approach adapted to a dualbeam focused ion beam and scanning electron microscope.

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In situ tensile tests on 75 nm diameter Cu nanowhiskers as well as compression tests on nanoporous Au micropillars fabricated using FIB annular milling are reported, the scientific question being the mechanical behaviour of nanosize materials. Both examples probably represent what will be the future of in situ mechanical tests using scanning electron microscopes. Conditions for experimentation Combination of the use of the ESEM and a Peltier stage with the development of specific detectors allows the possibility to control both specimen temperature and water pressure around the sample (Leary & Brydson, 2010). Water can be condensed or evaporated on the demand from the sample (Figure 3). This allows performing in situ experiments in a temperature-pressure domain that is reported on Figure 3a (dot zone). An easy to perform experiment, illustrated by a 6 images series, corresponding to the NaCl dissolution (during the increasing of the water pressure in the ESEM chamber and consecutive water condensation, at constant temperature) in water followed by the crystallization of NaCl (decrease of the water pressure) is reported on Figure 3b. This example corresponds to an "isothermal experiment". Another ways to work are to perform isobar experiments or to heat or cool a sample using a constant relative humidity (iso-RH experiments). These techniques allow the characterization of structural transitions of hydrated samples as a function of temperature (Bonnefond, 2011). Biology and soft matter applications This technique is particularly well adapted for the observation or experimentation on biological samples (Muscariello et al., 2005). Images of small and highly hydrated samples such as

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liposomes have been obtained by several authors (Perrie et al., 2007 ;Ruozi et al;) without any particular sample preparation. Perrie et al. (2007) have also been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses onto, or evaporates from, the sample in real-time. The data obtained provides an insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay for liposome formulation and stability (Perrie et al., 2010). However, Kirk et al. (2009) report that ESEM imaging of biological samples must remain combined with the classical techniques for sample preparation. Several works are specifically dedicated to in situ experimentation. Stabentheiner et al. (2010) state that "one unrivaled possibility of ESEM is the in situ investigation of dynamic processes that are impossible to access with CSEM where samples have to be fixed and processed". These authors have studied the anther opening that is a highly dynamic process involving several tissue layers and controlled tissue desiccation. This phenomenon can be observed because the sample is very stable under the ESEM conditions ( Figure 4). Another recent study is relative to the closure of stomatal pores by Mc Gregor & Donald (2010). Even if the possibility for experimentation on biological samples is clearly demonstrated, the authors outline the fact that the electron beam damages are important even at low accelerating voltage (Zheng et al., 2009). Another surprising example that can be reported is the direct observation of living acarids available online: in the movie, colonies of acarids

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are directly observed in the ESEM chamber under several conditions (FEI movie). Applications on cements Several works have been performed in order to study the reactivity of cement materials versus humidity. Hydration or dehydration (Sorgi & De Gennaro, 2007;Fonseca & Jennings, 2010;Camacho-Bragado et al., 2011) of phases have been followed and used to extract kinetic parameters (Montes-Hernandez, 2002 ;Montes & Swelling, 2005 ;Maison et al., 2009), as reported on Figure 5. In this work, the author uses ESEM image series to determine a three-step mechanism for bentonite aggregates evolution with relative humidity corresponding to an arrangement of particles followed by a particle swelling and a full destructuration. In SEM experiments are also used to characterize chemical reactivity (Camacho-Bragado et al., 2011). It has been recently used to characterize reaction of fly ash activated by sodium silicate by Duchene et al. (2010). These authors have determined very accurately the different steps of the reaction determining that the sodium silicate activator dissolves rapidly and begins to bond fly ash particles. Open porosity was observed and it was rapidly filled with gel as soon as the liquid phase is able to reach the ash particle. The importance of the liquid phase is underlined as a fluid transport medium permitting the activator to reach and react with the fly ash particles. The reaction products had a gel like morphology and no crystallized phase was observed. Hydration and dehydration experiments As previously reported for liposomes, new opportunities for the study of polyelectrolyte microcapsules versus their resistance to relative humidity and temperature

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modifications are opened and under consideration. The image series reported on Figure 6 clearly illustrate the possibility to image the native soft capsule at high relative humidity without any deformation. When decreasing the water pressure near the capsule, the object is deformed and do not shrink as observed when it is heated in water at temperature higher than 25°C (Basset et al., 2010). Thus, the walls of the object do not rearrange but collapse when submitted to a relative humidity decrease. Similar tests have been performed on self-organized metal-organic framework compounds (Bonnefond, 2011). According to the image series reported on Figure 7, when the water pressure decreases, the size of sample remains constant up to a given water pressure (i.e. relative humidity) and for a transition pressure, the sample size decreases regularly. This can be associated to a local reorganisation in the sample that corresponds to a water loss associated to the sample collapsing The enthalpy of water ordering in the sample can be derived from the recorded image series as reported by Sievers et al. The effect of dehydration on lamellar bones was also studied by in situ ESEM experiments (Utku et al., 2008). The obtained results indicate that dehydration affects the dimensions of lamellar bone in an anisotropic manner in longitudinal sections, whereas in transverse sections the extent of contraction is almost the same in both the radial and tangential directions. An original work on the heterogeneous ice nucleation on synthetic silver iodide, natural kaolinite and montmorillonite particles has been performed using the "increasing

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water pressure at constant temperature" (Zimmermann et al., 2007) in the temperature range of 250-270 K. Ice formation was related to the chemical composition of the particles. The obtained data are in very good agreement with previous ones obtained by diffusion chamber measurements (Figure 8). Characterization of surface wetting properties Characterization of the wetting properties of surfaces through the formation of microdroplets or nanodroplets is another important investigation field that can be explored using the ESEM. A recent review by Mendez-Vilas et al. (2009) has highlighted the main fundamental and applied results. Several strategies for the contact angle between water and the surface determination are reported (Stelmashenko et al., 2001;Stokes, 2001;Lau et al., 2003;Wei, 2004;Yu et al., 2006;Jung & Bhushan, 2008;Rykaczewski & Scott, 2011). The investigation of the hydrophobicity and/or hydrophilicity of a catalyst layer have been performed using ESEM for the first time by Yu et al. (2006). These authors have determined the micro-contact angle distribution as a function of the catalyst microstructure. Microdroplets growing and merging process was observed directly in the ESEM chamber by Lau et al. (2003). www.intechopen.com Using the Wet-STEM mode The development of the Wet-STEM by Bogner et al. (2005Bogner et al. ( , 2007 allows observing samples in the transmission mode in the ESEM chamber, and more particularly, it offers the possibility to image directly nanoparticles dispersed in a few micrometer thin water film (Bogner et al., 2008), emulsions or vesicles (Maraloiu et al., 2010), without removing the liquid surrounding the objects of interest. One must keep in mind

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that images with soft matter, and more generally sample sensitive to the electron beam are very hard to obtain. Nevertheless, this technique also opens new research fields using in situ experimentation that only begin to be explored for wettability or deliquescence studies. By combining Wet-STEM imaging with Monte-Carlo simulation (Figure 10), Barkay (2010) have studied the initial stages of water nanodroplet condensation over a nonhomogeneous holey thin film. This study has shown a preferred water droplet condensation over the residual water film areas in the holes and has provided corresponding droplet shape and contact angle. On a similar way, Wise et al. (2008) have studied water uptake by NaCl particles prior to deliquescence by varying the relative humidity in the Wet-STEM environment ( Figure 11). Development of specific materials for experimentation Several specific devices have been developed to characterize specific properties or reactions. Two of them will be shortly described below. Chen et al. (2011) have developed an experimental platform that can be used to investigate chemical reaction pathways, to monitor phase changes in electrodes or to investigate degradation effects in batteries. They have performed in situ experiment runs inside a scanning electron microscope (SEM) and tracked the morphology of an electrode including active and passive materials in real time. This work has been used to observe SnO 2 during lithium uptake and release inside a working battery electrode. www.intechopen.com Direct imaging of micro ink jets inside the ESEM chamber has been achieved using a specific device developed by Deponte et al. (2009), using a two-fluid

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stream consisting of a water inner core and a co-flowing outer gas sheath. ESEM images of water jets down to 700 nm diameter have been recorded. Details of the jet structure (the point of jet breakup, size and shape of the jet cone) can be measured. The authors conclude that ESEM imaging of liquid jets offers a valuable research tool for the study of aerosol production, combustion processes, ink-jet generation, and many other attributes of micro-and nanojet systems. Application domains of HT-(E)SEM Specific stages (and associated detectors) have been developed to heat samples up to 1500°C directly in the microscope chamber (Knowles & Evans, 1997;Gregori et al., 2001). The environmental scanning electron microscope (ESEM) equipped with this heating stage is an excellent tool for the in situ and continuous observation of system modifications involved by temperature. It allows recording image series of the morphological changes of a sample during a heat treatment with both high magnification and high depth of focus. The experiments can be carried out to observe the influence of all these parameters on the studied phenomenon under various conditions (heating rates, atmosphere compositions, variable pressure, final temperature and heating time). Images have been recorded up to 1400°C, with a decrease of the image resolution when the sample temperature increases (Podor et al., 2012). It is possible to work under vacuum (classical SEM) or under controlled atmosphere (H 2 O, O 2 , He+H 2 , N 2 , air...). Different types of studies have been reported, relative to corrosion of metals (Jonsson et

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al., 2011), oxidation of metals (Schmid et al., 2001a(Schmid et al., , 2001bOquab & Monceau, 2001 ;Schmid et al., 2002 ;Abolhassani et al., 2003 ;Reichmann et al., 2008 ;Jonsson et al., 2009 ;Mège-Revil et al., 2009 ;Quémarda et al., 2009 ;Delehouzé et al., 2011), reactivity at high temperature (Maroni et al., 1999 ;Boucetta et al., 2010), phase changes (Fischer et al., 2004 ;Hung et al., 2007 ;), hydrogen desorption (Beattie et al., , 2011, redox reactions (Klemensø et al., 2006), microstructural modifications (Bestmann et al., 2005 ;Fielden, 2005 ;Yang, 2010), magnetic properties (Reichmann et al., 2011), sintering (Sample et al., 1996 ;Srinivasan, 2002 ;Marzagui & Cutard, 2004 ;Smith et al., 2006 ;Subramaniam, 2006 ;Courtois et al., 2011 ;Joly-Pottuz et al., 2011 ;Podor et al., 2012), thermal decomposition (Gualtieri et al., 2008 ;Claparède et al., 2011 ;Goodrich & Lattimer, 2011 ;Hingant et al., 2011), crystallisation (Gomez et al., 2009 in melts (Imaizumi et al., 2003 ;Hillers et al., 2007) and study of self-repairing -self-healingproperties of materials (Wilson & Case, 1997 ;Coillot et al., 2010aCoillot et al., , 2010bCoillot et al., , 2011) … Even if numerous researchers are invested in HT-ESEM, only few of them have been successful in pursuing dynamic experiments at temperatures higher than 1100°C. Two recent studies report experiments performed at T=1350°C (Subramaniam, 2005) and 1450°C (Gregori et al., 2002). However, the resolution of the images remains poor (more than 1µm) mainly due to water cooling induced vibrations. Furthermore, the precision on the measure of the sample temperature remains poor (temperature differences up to

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150°C with the expected temperature are sometimes measured). A recent device has been proposed by Podor et al. (2011) to overcome this difficulty. A complete review specifically dedicated to in situ high temperature experimentation in the ESEM will be available soon. Several examples of in situ studies performed at high www.intechopen.com temperature in the ESEM chamber will be reported below, on the basis of original data acquired in our laboratory. Investigation of the crystallization behaviour in silicate melts The crystal growth and morphology during isothermal heating of glass melts can be directly observed using the hot stage associated with the ESEM. The image series reported on Figure 12 have been recorded during 10 minutes while heating the borosilicate melt sample isothermally at T=740°C. The development of large crystals in the melt rapidly yields to the complete crystallization of the melt. The crystal morphology presents cells filled with a second phase and the crystal formation yields to the deformation of the sample surface. Hillers et al. (2007) have used such data to quantify the variation of crystal length with time. They have established that the growth is only linear during the first minutes; afterward the growth rate decreases progressively with time. This technique can also be used to determine the temperature of formation of the first crystals at the melt surface and to observe their formation. In the case of glass-ceramics, the density of nuclei as well as their size and shape development can be directly observed and used for crystallization kinetic determination (Vigouroux et al., 2011,

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in prep). Fig. 12. Growth of crystals in a borosilicate melt during 10 minutes isothermal heat treatment at 740°C observed using the hot stage associated with the ESEM. Decomposition of compounds In situ thermal decomposition of composites, oxalates, oxides have been reported by several authors. Images of the heat treatment of a mixed uranium-cerium oxalate grain from 25°C to 1235°C are gathered on Figure 13. Morphological changes with temperature are directly linked with the oxalate decomposition as stated by Hingant et al. (2011) in the temperature range 25-500°C. The sample shrinkage observed when T>500°C is probably related with the first stage of the sintering process -i.e. beginning of bond formation between the nanograins and with the oxide grain growth (that can not be directly observed at this stage by HT-ESEM, but that is confirmed by X-Ray diffraction). Such a process has also been recently reported by Claparede et al. (2011) andJoly-Pottuz et al. (2011). Fig. 13. Decomposition of a uranium-cerium mixed oxalate observed during in situ heating in the ESEM chamber and relative size and shrinkage modifications. Study of sintering and grain growth Several studies are relative to the sintering and grain growth processes in metals and ceramics. Depending on the system, the experiments have been performed in the temperature range 300-1450°C. The main interest of these studies is the possibility of direct observation of the individual grain behaviour during heat treatment. The example that is reported on Figure 14a corresponds to the heat treatment of the grain decomposed in situ ( Figure 13). The image

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resolution is high enough to observe the nanograins growth inside the square plate agglomerate. Consequently, relative shrinkage and average grain diameter are extracted by image processing (Figure 14b). Assuming that the final density of the agglomerate is 99%, the sintering map is directly derived from these experimental data ( Figure 14c). Thus, in situ sintering experiments can allow the establishment of the trajectories of theoretical sintering. Such data have never been already reported in previous studies, mainly due to the poor resolution of the recorded images. The effect of the electron beam on sintering is controversy. Indeed, Popma (2002) noted that a local sintering stop was achieved by focusing the electron beam at a certain position during the in situ sintering experiments in the ESEM (performed on ZrO 2 nanolayers). On the contrary, Courtois et al (2011) performed experiments on the sintering of a lead phosphovanadate and concluded that the electric current induced by the electron beam was found to reduce the effective temperature of sintering by 50 to 150°C as well as to accelerate the kinetics of shrinkage of a cluster composed of sub-micrometric grains of material. Such effects were not evidenced in our study: the local sintering on sample surface zones that were not observed (i.e. exposed to the electron beam) was identical to the local sintering determined on the observed zone. Conclusions and perspectives In situ scanning electron microscopy experimentation, that is generally associated with the use of the ESEM, allows the study of very different problems, the main limit being the availability

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of specific devices. Torres & Ramirez (2011) have written the best conclusion indicating that "the new generation of SEMs shows innovative hardware and software solutions that result in improved performance. This progress has turned the SEM into an extraordinary tool to develop more complex and realistic in situ experiments, achieving even at the subnanometer scale". In the near future, new SEM imaging modes, nanomanipulation www.intechopen.com and nanofabrication technologies (Miller & Russell, 2007 ;Romano-Rodriguez & Hernandez-Ramirez, 2007 ;Wich et al., 2011) will make possible to replicate more closely the conditions as the ones associated to the problems to be treated. In situ ESEM will probably be used to overcome technical and fundamental challenges in many scientific domains. The recent developments of a high temperature stage in the FIB (Fielden, 2008), a new tomography mode in the ESEM (Jornsanoh et al., 2011) and of the atmospheric scanning electron microscope (Nishiyama et al, 2010 ;Suga et al, 2011) can be cited as examples for this future. Today, an individual would be hard-pressed to find any science field that does not employ methods and instruments based on the use of fine focused electron and ion beams. Well instrumented and supplemented with advanced methods and techniques, SEMs provide possibilities not only of surface imaging but quantitative measurement of object topologies, local electrophysical characteristics of semiconductor structures and performing elemental analysis. Moreover, a fine focused e-beam is widely used for the creation of micro and nanostructures. The book's approach covers both theoretical and practical issues related to scanning electron microscopy. The book

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has 41 chapters, divided into six sections: Instrumentation, Methodology, Biology, Medicine, Material Science, Nanostructured Materials for Electronic Industry, Thin Films, Membranes, Ceramic, Geoscience, and Mineralogy. Each chapter, written by different authors, is a complete work which presupposes that readers have some background knowledge on the subject.

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Computational prediction of protein subdomain stability in MYBPC3 enables clinical risk stratification in hypertrophic cardiomyopathy and enhances variant interpretation Purpose Variants in MYBPC3 causing loss of function are the most common cause of hypertrophic cardiomyopathy (HCM). However, a substantial number of patients carry missense variants of uncertain significance (VUS) in MYBPC3. We hypothesize that a structural-based algorithm, STRUM, which estimates the effect of missense variants on protein folding, will identify a subgroup of HCM patients with a MYBPC3 VUS associated with increased clinical risk. Methods Among 7,963 patients in the multicenter Sarcomeric Human Cardiomyopathy Registry (SHaRe), 120 unique missense VUS in MYBPC3 were identified. Variants were evaluated for their effect on subdomain folding and a stratified time-to-event analysis for an overall composite endpoint (first occurrence of ventricular arrhythmia, heart failure, all-cause mortality, atrial fibrillation, and stroke) was performed for patients with HCM and a MYBPC3 missense VUS. Results We demonstrated that patients carrying a MYBPC3 VUS predicted to cause subdomain misfolding (STRUM+, ΔΔG ≤ −1.2 kcal/mol) exhibited a higher rate of adverse events compared with those with a STRUM- VUS (hazard ratio = 2.29, P = 0.0282). In silico saturation mutagenesis of MYBPC3 identified 4,943/23,427 (21%) missense variants that were predicted to cause subdomain misfolding. Conclusion STRUM identifies patients with HCM and a MYBPC3 VUS who may be at higher clinical risk and provides supportive evidence for pathogenicity. INTRODUCTION Genetic variant interpretation is an ongoing challenge in clinical medicine, particularly when the gene of interest lacks robust functional assays. 1,2 A variety of computational algorithms have

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been developed to predict variant pathogenicity, but their sensitivity and specificity are often poor, particularly when applied broadly across different diseases and different genes. 1,3 Loss-offunction (LoF) pathogenic variants are common, 1,4,5 resulting from either frameshift or nonsense variants creating a premature stop codon, splice errors, disruption of enzymatic activity, alteration of protein-protein interactions, or protein misfolding. 1,6,7 Recognizing a common mechanism by which variants in a particular gene lead to LoF can inform the development of gene-specific computational algorithms to more accurately predict pathogenicity among variants that cannot be confidently classified based on clinical and family data alone. 6,7 Herein we focus on MYBPC3 (encoding the protein, cardiac myosin binding protein C, or MyBP-C). Pathogenic variants in MYBPC3 account for~50% of patients with sarcomeric hypertrophic cardiomyopathy (HCM), 8,9 and are inherited in an autosomal dominant fashion (OMIM 115197). Patients with HCM can experience a variety of adverse clinical outcomes, including outflow tract obstruction, arrhythmias, heart failure, and sudden cardiac death. 8 Genetic variants in MYBPC3 consist of both truncating and nontruncating types. Rarely found in healthy populations, truncating MYBPC3 variants result in a premature stop codon and cause HCM through complete LoF and haploinsufficiency at the transcript and protein level. [10][11][12][13] Thus, interpretation of these truncating variants as pathogenic is straightforward. 14 However, the interpretation of missense variants within MYBPC3 presents a major challenge. Single amino acid substitutions (missense variants) are found commonly in healthy populations. Further, since missense variants do not disrupt the reading frame, protein function may be tolerant to these minor sequence

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changes. Thus, many missense variants lack sufficient evidence to be classified as either pathogenic or benign and are classified as variants of uncertain significance (VUS). 14,15 While identifying pathogenic variants allows for predictive genetic testing in at-risk relatives, 16 a VUS is not clinically actionable and may lead to misinterpretation by clinicians and patients. 17 Identification of a pathogenic sarcomere genetic variant for HCM also has important prognostic implications. Patients with HCM and a pathogenic sarcomere variant (sarcomeric HCM) have a higher risk of adverse clinical outcomes compared with those without a sarcomere gene variant (nonsarcomeric HCM). 8,18 Patients carrying a sarcomere gene VUS, on average, exhibit an intermediate risk of adverse events, 8 most likely because VUS represent a mixed pool of pathogenic and benign variants that cannot be parsed on the basis of clinical and genetic data alone. Because LoF is an established mechanism for pathogenic variants in MYBPC3, we hypothesized that applying a computational approach, called STRUM, 19-21 that incorporates both sequencebased and structure-based algorithms to missense MYBPC3 VUS will identify those variants that result in protein subdomain misfolding (STRUM+), thereby supporting pathogenicity and improving variant interpretation. We further predict that this approach will identify a subpopulation of patients with HCM and a STRUM+ MYBPC3 missense VUS who are at risk for adverse clinical outcomes, at a frequency similar to patients with HCM carrying known pathogenic variants. Sarcomeric Human Cardiomyopathy Registry (SHaRe) data extraction and MYBPC3 variant classification The generation of the centralized SHaRe database has been previously described. 8 Data were exported

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from quarter 1 of 2019. Inclusion criteria included a site-designated diagnosis of HCM using standard diagnostic criteria. 8 SHaRe nontruncating MYBPC3 missense variants (Tables S1, S2) were classified as previously reported 14 in accordance with American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) joint guidelines, leveraging available clinical and experimental data. 3,8,9,14,22,23 Known splice variants are classified as truncating. Since variants in MYBPC3 present in gnomAD with allele frequencies of >4E-05 and absent in SHaRe are unlikely to be independently pathogenic for HCM, these variants were included in our list of benign MYBPC3 variants. 14 More details regarding variant interpretation are provided within the Supplemental materials. It has previously been shown that patients carrying pathogenic nontruncating variants exhibit similar clinical outcomes to those carrying truncating MYBPC3 variants. 14 Thus, a reference population including previously adjudicated truncating and nontruncating MYBPC3 pathogenic/ likely pathogenic (pathogenic) variants (MYBPC3-path-all) was used. A second reference population included patients with HCM who underwent genetic testing and were negative for sarcomere variants Sarc-. 8 Computational structural and protein folding stability predictive modeling MyBP-C is made up of immunoglobulin and fibronectin subdomains (C0-C10) (NM_000256.3, NP_000247.2). For MYBPC3 missense variants we utilized STRUM to calculate the effect of the missense variant on the Gibbs free energy of local subdomain folding (ΔΔG) 19 (Table S3). A negative ΔΔG value indicates the degree of reduced folding energy (kcal/mol) relative to the wild-type subdomain, or folding destabilization. 19 Previous experimental validation of this algorithm compared STRUM predictions to 3,421 experimentally tested variants

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from 150 proteins and demonstrated a Pearson's correlation coefficient of 0.79 and root mean square error of prediction of 1.2 kcal/mol. 19 Thus, a value of ΔΔG ≤ −1.2 kcal/mol was defined as the cutoff for destabilizing (deleterious) variants. Further details regarding STRUM analysis and structural models are provided within the Supplemental Materials ( Figure S1-S3, Table S3). Computational sequence-based variant analysis (PolyPhen-2, SIFT, CardioBoost) We compared the STRUM prediction for MYBPC3 missense variants with a sequence-based algorithm embedded in STRUM (SIFT). 24,25 We also analyzed these variants with PolyPhen-2 (HumVar database), another sequence based algorithm. 26 Finally, we compared our result with those obtained using CardioBoost, which is a disease specific machine learning classifier to predict pathogenicity of rare missense variants in genes associated with cardiomyopathies and arrhythmias. 6 CardioBoost relies on minor allele frequency, whereas STRUM does not. Clinical outcomes analysis Only patients with HCM carrying a single MYBPC3 missense VUS were included in clinical outcomes analyses to avoid confounding from cases with multiple gene variants. 27 Comparisons using time-to-event analysis were made between variants predicted to be deleterious (STRUM+, ΔΔG ≤ −1.2 kcal/mol) and those predicted to be nondeleterious. The primary outcome was an overall composite previously defined as the first occurrence of any component of the ventricular arrhythmia composite, heart failure composite (without inclusions of LV ejection fraction), allcause mortality, atrial fibrillation (AF), or stroke. 8 Results were compared with reference populations MYBPC3-path-all and Sarc-. A secondary analysis of a heart failure composite, ventricular arrhythmia composite, and atrial fibrillation was also performed.

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Finally, a secondary analysis using alternative computational algorithms (SIFT, PolyPhen-2, CardioBoost) was performed. Composite outcomes are defined in more detail in the Supplemental materials. Statistical analysis Data presented as mean ± standard deviation were analyzed by t-test for two groups or analysis of variance (ANOVA) for >2 groups with Tukey's post hoc test for multiple comparisons. Data presented as frequency were analyzed by a chi-square test. Odds ratio (with 95% confidence interval [CI]), specificity, and sensitivity were calculated to evaluate the association between computational prediction algorithms and known pathogenic/ likely pathogenic (pathogenic) or benign/likely benign (benign) variants (further details provided in supplemental materials). Primary and secondary clinical outcomes were analyzed by the Kaplan-Meier method from time of birth. Analysis from time of birth is appropriate given that the genetic variant is present from birth and variability in time to, and reason for, clinical presentation could confound the results if time from diagnosis were used. Patients who did not have the outcome of interest were censored at the time of their last recorded follow-up in SHaRe. Comparison between curves was performed using Log-rank Mantel-Cox test with p values of <0.05 considered statistically significant. Median event free survival and hazard ratio (Mantel-Haenszel) are also reported. Statistical analyses were performed using GraphPad Prism software (San Diego, CA). RESULTS Patients with HCM and a MYBPC3 missense VUS predicted to disrupt subdomain folding (STRUM+) exhibit a higher incidence of adverse clinical outcomes We began by evaluating all MYBPC3 missense VUS within SHaRe using STRUM. MYBPC3 VUS exhibited a mean ΔΔG of

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−0.73 ± 1.06 kcal/mol ( Figure S4). Of 120 unique MYBPC3 missense VUS, 34 (28%) were predicted to cause subdomain misfolding with ΔΔG values ≤ −1.2 kcal/mol (deleterious) (Table S2). Next, we evaluated clinical characteristics and outcomes in patients with HCM and a single missense MYBPC3 VUS predicted to disrupt subdomain folding (STRUM+) compared with patients carrying a VUS not predicted to disrupt subdomain folding (STRUM-). For this analysis, we included only patients who carried a single VUS within MYBPC3, and excluded patients who carried a second pathogenic variant or variant of uncertain significance (N = 105). Patients with a STRUM+ versus STRUM-MYBPC3 VUS exhibited similar clinical characteristics including body mass index (BMI), gender, ancestry, age at diagnosis, wall thickness, ejection fraction, and left ventricular outflow tract obstruction (Table 1). We observed that patients carrying a STRUM+ VUS experienced higher rates of adverse events compared with patients carrying a STRUM-VUS ( Fig. 1, hazard ratio 2.3, p = 0.03). Furthermore, patients carrying a STRUM+ VUS exhibited a similar rate of adverse clinical events compared with patients carrying a pathogenic variant (MYBPC3-Path-all). Conversely, patients carrying STRUM-VUS exhibited a lower frequency of outcomes, similar to Sarc-patients (Fig. 2). There were no statistically significant differences between groups for the individual component outcomes, including ventricular arrhythmias, heart failure, or atrial fibrillation ( Figure S5). STRUM exhibits improved specificity over established sequencebased prediction algorithms and improved sensitivity when combined with CardioBoost To determine the sensitivity and specificity of STRUM to differentiate pathogenic from benign variants within MYBPC3 we performed STRUM analysis

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on all known pathogenic missense variants within ShaRe (n = 19) and known missense benign variants within SHaRe and gnomAD (n = 110, Table S1, Fig. 3a). These variants were present in 412 patients with HCM within the SHaRe registry. MYBPC3 benign variants exhibited a mean ΔΔG of −0.31 ± 0.60 kcal/mol, which was significantly higher than MYBPC3 VUS (ΔΔG of −0.73 ± 1.06 kcal/mol, p = 0.005) ( Figure S4) and MYBPC3 pathogenic variants (mean ΔΔG of −1.00 ± 1.08 kcal/mol, p = 0.016) (Fig. 3a). We found that variants predicted to be deleterious by STRUM were more likely to be pathogenic variants (odds ratio [OR] 5.9, 95% CI 1.8-19.6) (Fig. 3c). Only nine additional unique nontruncating MYBPC3 variants were designated as pathogenic and/or likely pathogenic within ClinVar. However, all of these variants had a single submission and a review status of 0-1/4 criteria provided. By modern standards, these variants would be reclassified as VUS and were therefore not included in our analysis. Algorithms that were purely sequence-based achieved greater sensitivity but performed inferiorly to STRUM in regard to specificity. STRUM exhibited a 93% specificity for benign variants and PolyPhen-2 and SIFT exhibited a specificity of 62% (OR 4.5, 95% 1.5-13.5) and 54% (OR 1.3, 95% CI 0.5-3.4) respectively (Fig. 3c, Figure S6). Additionally, variant interpretation by SIFT or PolyPhen-2 did not stratify patients carrying a MYBPC3 VUS for clinical adverse outcomes ( Figure S6). In comparison, CardioBoost demonstrated a specificity of 98% (OR 42.3, CI 8.0-223.6) (Fig. 3, Table S1). For pathogenic variants, CardioBoost

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demonstrated a sensitivity of 47%. Interestingly, there was limited overlap among known pathogenic variants predicted to be deleterious by STRUM and those predicted to be deleterious by CardioBoost, making the two algorithms complementary (Table S1). Combining these algorithms to classify any variant predicted to be deleterious by CardioBoost or STRUM as pathogenic maintained a high specificity of 93% and improved sensitivity to 63% (Fig. 3c). When examining patients with HCM and a MYBPC3 missense VUS, STRUM identified a larger number of MYBPC3 VUS as deleterious. Only 16 of 39 (41%) patients with a STRUM+ MYBPC3 VUS were also identified as CardioBoost+. Just three additional patients were uniquely identified as CardioBoost+ (Table S2). While there is a trend toward a higher rate of adverse clinical events in patients with HCM and a CardioBoost+ MYBPC3 VUS, this difference was not statistically significant (Fig. 3d) to the sarcomere and were rapidly degraded within primary cardiomyocytes. 14 Consistent with these experimental findings, pathogenic C10 domain variants are uniformly predicted to destabilize protein folding (ΔΔG of −2.89 and −1.45 kcal/mol respectively) (Fig. 4). Conversely, of the pathogenic MYBPC3 variants not predicted to be deleterious by STRUM (Fig. 3), a large number were localized within the C3 domain (Fig. 3a, open circles; 7/13) and exhibited a mean ΔΔG −0.32 kcal/mol, (range −0.93 to 0.04). A large number of known pathogenic variants cluster within the C3 domain near a surface-exposed flexible linker (Fig. 4). 15 Thus, these variants would be predicted to alter electrostatic protein-protein interactions but would not be expected to disrupt

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subdomain folding. This result is consistent with prior experimental and structural characterization data of these C3 pathogenic variants. Arg495Gln, Arg502Trp, and Phe503Leu incorporate normally into the sarcomere and have protein half lives that are indistinguishable from wild-type MyBP-C within primary cardiomyocytes. 14 Further, the NMR structure of the MYBPC3 Arg502Trp C3 domain reveals preserved subdomain folding. 28 While C3 and C10 pathogenic variants have a narrow range of ΔΔG values, ΔΔG predictions for C6 pathogenic variants vary from −2.33 to 0.04 (mean ΔΔG −1.11). We previously examined two C6 domain variants, Arg810His and Trp792Arg, and found that they incorporate normally into the sarcomere and exhibit normal protein half lives in primary cardiomyocytes. 14 However, both of these variants were predicted to destabilize subdomain folding by STRUM, exhibiting values near the cutoff: Arg810His (ΔΔG −1.22 kcal/mol), Trp792Arg (ΔΔG −1.28 kcal/mol). They are also predicted to be pathogenic by CardioBoost (Table S2). These observations suggest that a subset of pathogenic variants mildly disrupt subdomain folding without causing complete destabilization of MyBP-C. Subdomain destabilization in these cases could interfere with protein-protein interactions or MyBP-C conformational dynamics. In silico saturation mutagenesis of MYBPC3 identified 4,943 missense variants predicted to cause subdomain misfolding Only a subset of amino acid substitutions has been observed in patients with HCM and are cataloged in publicly available databases, such as ClinVar. However, previously unreported variants frequently arise in probands with HCM who undergo clinical genetic testing. 29 Thus, we performed STRUM on all possible MYBPC3 single amino acid substitutions (in silico mutagenesis) to develop a

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compendium of STRUM+ variants that may be useful for the research and clinical community. We found that 4,943 of 24,665 (20%) amino acid substitutions were predicted to disrupt subdomain folding ( Figure S6, Tables S4, S5). DISCUSSION Clinical risk stratification has been a cornerstone of clinical HCM management. It is well-established that patients with sarcomeric HCM have a higher rate of adverse clinical outcomes compared with nonsarcomeric HCM, enabling the incorporation of genetic data into clinical risk stratification in HCM. 8,18 Yet, refinement of clinical risk for patients with a VUS remains an ongoing challenge for clinicians. 1,5 We have identified a subpopulation of patients with a MYBPC3 missense VUS that are predicted to disrupt subdomain protein folding (STRUM+) who exhibit clinical outcomes indistinguishable from patients with a pathogenic MYBPC3 variant. Conversely, patients carrying a MYBPC3 VUS not predicted to affect subdomain folding (STRUM−), exhibit a lower prevalence of adverse clinical outcomes similar to patients with nonsarcomeric HCM. Although the methodology of parsing these variants is different for MYBPC3 because of differing underlying mechanisms, these findings are analogous to a recent study in MYH7 in which patients with HCM carrying VUS that were located within the interacting heads motif had a higher rate of adverse clinical outcomes compared with patients carrying VUS that were outside of this motif. 30 These studies together suggest that VUS in sarcomere genes are primarily an admixture of pathogenic and benign variants. So, while patients with HCM carrying sarcomere gene VUS as a whole exhibit a prevalence of clinical outcomes that

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are intermediate between patients with or without pathogenic sarcomere variants, 8 a computational approach specifically leveraging the pathogenic mechanism of MYBPC3 has enabled the identification of higher risk subpopulation that exhibit clinical outcomes similar to sarcomeric HCM and a lower risk subpopulation that exhibit clinical outcomes similar to nonsarcomeric HCM. While computational prediction should not be exclusively relied on to assign pathogenicity of a variant or risk stratify an individual patient, STRUM could be incorporated in an additive manner with other methods for variant adjudication to prioritize variants that warrant further investigation. Given that novel MYBPC3 variants are frequently identified by genetic testing of probands with HCM, 29 we completed an in silico "saturation mutagenesis" of MYBPC3 compiling a complete list of STRUM+ variants. Excluding known pathogenic or benign variants, we estimate that~0.097% (1/1,033) individuals within gnomAD carry a MYBPC3 variant predicted to cause subdomain misfolding by STRUM. STRUM+ MYBPC3 VUS identified in patients with HCM should be prioritized for additional clinical and experimental investigation. Specifically, functional experimental studies to evaluate the direct effects of MYBPC3 VUS on protein stability, folding, and localization, as we have done previously for a subset of pathogenic variants, 14 19 analysis for MYBPC3 pathogenic and benign variants within C3, C6, and C10 are shown, with mean and SEM for each group depicted. Graph is labeled to indicate variants predicted to be deleterious. When known benign missense variants were evaluated by STRUM, 102 of 110 variants were correctly predicted, with an overall specificity of 93%. However, for known pathogenic variants, only

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7 of 19 were predicted to alter subdomain folding by STRUM, yielding a sensitivity of 32%. This lower sensitivity was in large part explained by a known cluster of pathogenic variants within C3. 15 None of the seven known pathogenic variants in C3 had a ΔΔG value below the threshold of −1.2 kcal/mol. This is consistent with experimental data that demonstrate C3 variants localize normally to the sarcomere and exhibit protein half lives similar to wild-type MyBP-C. Additionally, an NMR structure of Arg502Trp demonstrates that this variant does not disrupt subdomain folding but rather is more likely to alter protein-protein interactions. 14,28 In contrast, MyBP-C pathogenic variants in C10, predicted by STRUM to cause subdomain misfolding, fail to localize to the sarcomere and are rapidly degraded. 14 These experimental results support the accuracy of STRUM predictions for subdomain misfolding. Further, they highlight that STRUM is only predictive of pathogenicity for variants that significantly alter protein folding as their primary mechanism. Thus, a ΔΔG value of > −1.2 kcal/mol does not exclude pathogenicity for variants that cause loss or gain-offunction through an alternate mechanism such as alternative splicing or altered protein-protein interactions. STRUM is best applied to VUS after other clinical, computational, and experimental criteria for variant adjudication have been implemented. For example, MYBPC3 pathogenic variants that lead to LoF by mechanisms other than subdomain misfolding have previously been well characterized and defined as pathogenic, including splice variants 14,22,23 and the cluster of pathogenic variants within C3 (aa.485-503) 15,28,31 discussed above. STRUM performed superiorly to sequence based

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algorithms alone, such as SIFT and PolyPhen-2, which each had lower specificity and were unable to clinically risk stratify patients with HCM and a MYBPC3 missense VUS. Compared with using each method independently, combining STRUM and CardioBoost improved sensitivity for identifying known pathogenic variants to 63% while maintaining a specificity for known benign variants of 93%. CardioBoost supported pathogenicity for four missense VUS that were STRUM -but only predicted pathogenicity for 12/34 of MYBPC3 STRUM+ VUS. This result highlights the added utility of STRUM to identify a subset of VUS within MYBPC3 that result in local subdomain misfolding leading to allelic LoF and have a high probability of being pathogenic. Because CardioBoost and STRUM are complementary and have high specificity, we would propose that the ACMG/AMP PP3 criteria, where multiple lines of computational evidence support a deleterious effect of a variant, could be applied when one or both algorithms predict pathogenicity. Conversely, because of relatively limited sensitivity for each algorithm independently, we would propose that the BP4 criteria, where multiple lines of computational evidence support no impact of the variant, be applied only if both algorithms predict that a variant is nonpathogenic. Although this study was limited by a moderate sample size of 105 patients with HCM, the comprehensive variant adjudication in SHaRe enabled strict inclusion of patients carrying a single VUS within MYBPC3 to clearly discriminate genetic-clinical correlates in this population. This approach enabled us to discern a difference in a composite of adverse clinical outcomes between patients with STRUM+ and STRUM-variants. However, the sample

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size was insufficient for detecting differences in individual outcomes, such as arrhythmias or heart failure, and did not provide sufficient power to correct for other risk predictors. The approach of using STRUM as an adjunctive tool for decision making may also be applicable to other genes for which LoF is a pathogenic mechanism. Approximately 50% of disease associated variants within Human Gene Mutation Database are truncating variants predicted to result in LoF. 11 These genes, like MYBPC3, also have missense VUS that may be evaluated for protein misfolding using STRUM. For example, there are several causal genes for hypertrophic, dilated, and arrhythmogenic cardiomyopathies with truncating pathogenic variants, including lamin A/C, desmoplakin, and plakophilin 2, Titin, and phospholamban. 11,32 This approach is best suited for nonenzymatic proteins where high-quality structural modeling can be performed, and for which the primary pathogenic mechanism has been established to be LoF. Conclusions We show that the computational algorithm STRUM, that predicts protein structure stability in response to missense variation, enables identification of patients carrying a MYBPC3 VUS who may be at higher clinical risk of adverse events. This approach also provides supportive evidence for pathogenicity, prioritizing variants for functional experimental studies and clinical familial segregation to improve MYBPC3 variant adjudication. Finally, STRUM may be broadly applicable to variants in other genes for which LoF is an established mechanism. DATA AVAILABILITY De-identified data will be made available by request to the authors.

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Serum Soluble (Pro)renin Receptor Level as a Prognostic Factor in Patients Undergoing Maintenance Hemodialysis Abstract Background: The (pro)renin receptor [(P)RR)] is a multifunctional protein with roles in angiotensin II-dependent and -independent intracellular cell signaling and as an adaptor protein between the Wnt receptor complex and vacuolar proton-translocating adenosine triphosphatase. The (P)RR is cleaved to generate soluble (P)RR [s(P)RR], reflecting the status of the tissue renin-angiotensin system and/or activity of the (P)RR. Patients undergoing hemodialysis (HD) have poor prognosis because of the increased prevalence of cardiovascular and malignant diseases. This study was conducted to investigate whether the s(P)RR level is associated with new onset of cardiovascular events or malignant diseases and prognosis in patients undergoing HD. Methods: A total of 258 patients undergoing maintenance HD who were enrolled in our cohort study investigating the relationships between serum s(P)RR levels and background factors were prospectively followed up for 60 months. We investigated the relationships between s(P)RR levels and new onset of cardiovascular events or malignant diseases and mortality during the follow-up period. Results: The cumulative incidence of new onset of cardiovascular events (P = 0.009) and cardiovascular deaths (P < 0.001), but not of malignant diseases, was significantly greater in patients with higher serum s(P)RR level (≥ 29.8 ng/ml) than in those with lower s(P)RR level (< 29.8 ng/ml). A high serum s(P)RR level was independently correlated with cardiovascular mortality (P = 0.046). Conclusions: These data showed that the serum s(P)RR level is associated with cardiovascular events and mortality, suggesting that the serum s(P)RR level could be used

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as a biomarker for selecting patients requiring intensive care. Introduction The (pro)renin receptor [(P)RR], a speci c receptor for renin and prorenin, consists of 350 amino acids with a single transmembrane domain and preferentially binds to renin and prorenin. 1It is widely expressed in various organs, such as the brain, heart, and kidneys. 2,3Non-proteolytic renin activation by the binding of prorenin to the extracellular domain of (P)RR 4 accelerates the conversion of angiotensinogen to angiotensin (Ang) I.This mechanism has been proposed as a source of renin activity in the tissue renin-angiotensin system. 1 The soluble form of PRR[s(P)RR] is generated by intracellular cleavage by processing enzymes and is secreted into the extracellular space and found in the blood.These ndings suggest that s(P)RR can serve as a biomarker that re ects the status of the tissue RAS and/or (P)RR activity. 5,6Moreover, (P)RR is a multi-functioning protein that allows local production of Ang I from angiotensinogen and induces intracellular signals independent of RAS activation. It was recently discovered that (P)RR also functions as an adaptor protein between the Wnt receptor complex and vacuolar proton-translocating adenosine triphosphatase (V-ATPase). 7][10] Recently, accumulating evidence has revealed that overexpression of (P)RR, which may contribute to cancer initiation and progression, has been observed in various cancers including breast carcinoma, 11 pancreatic ductal adenocarcinoma, 12 glioma, 13 and aldosterone-producing adenoma. 14Patients undergoing hemodialysis (HD) have a poor prognosis because of the increased prevalence of cardiovascular diseases (CVD) in this population. 15,16Patients with heart failures have signi cantly higher plasma s(P)RR levels than control subjects.

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17We previously reported that the serum s(P)RR level was associated with arteriosclerosis, independent of other risk factors, in patients undergoing HD, 18 and the high serum s(P)RR level was associated with increases in brain natriuretic peptide (BNP), a marker of left ventricular dysfunction, 19 independent of other risk factors.These suggested that the increased expression of (P)RR may be associated with the progression of heart failure in patients undergoing HD. 20 Furthermore, it has been reported that patients undergoing HD are at a high risk for malignant diseases. 21,22However, whether the blood s(P)RR levels are associated with the development of cardiovascular events or malignant diseases in patients undergoing HD has not been reported.Furthermore, it remains undetermined whether the blood s(P)RR level is related to total deaths and/or cardiovascular deaths in patients undergoing HD.On the basis of these background ndings and unresolved questions, the present study aimed to investigate whether the serum s(P)RR level was associated with new-onset cardiovascular events or malignant diseases and with prognosis in patients undergoing HD during a follow-up period of 60 months. Study subjects The participants were outpatients on maintenance HD at the Kadoma Keijinnkai Clinic, Neyagawa Keijinnkai Clinic, and Moriguchi Keijinnkai Clinic in Osaka Prefecture, Japan, in April 2014.All three clinics are a liated with the Moriguchi Keijinkai Hospital, Osaka, Japan.This study was approved by the ethics committee of Tokyo Women's Medical University (approval number: 2703) and was conducted in accordance with principles of the 1975 Declaration of Helsinki, as revised in 2013.All patients were enrolled after they provided written informed consent.A

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total of 258 patients undergoing maintenance HD, who were enrolled in our previous cohort study that investigated the relationships between serum s(P)RR levels and background factors, 18 were prospectively followed up for 60 months.Each patient underwent HD therapy thrice a week for 3-4 hours at the same time each day. Background factors At the start of this study, we collected information on the study population, including age, sex, body mass index (BMI), primary disease (the presence or absence of diabetes mellitus), duration of HD, smoking status, past history of cardiovascular events and malignant diseases, urine volume (≥ 0 ml/day), and consumption of selected medications.BMI was calculated as follows: BMI = {post-dialysis body weight (kg) / [height (m)] 2 } × 100.Five systolic blood pressure (SBP) values were measured on the 1st dialysis day of the week: the rst was SBP at the start of dialysis; the second and third were the highest and lowest SBP during dialysis, respectively; the fourth was the difference between the highest and lowest values; the fth was SBP at the end of dialysis.The post-dialysis cardiothoracic ratio (CTR) values were obtained on the 1st dialysis day of the week.The normalized dialysis dose (Kt/V) was calculated on the 1st dialysis day of the week using the following equation, i.e., the formula of Daugirdas 23 : Kt/V = -Ln {[(post-dialysis value of blood urea nitrogen (BUN)/pre-dialysis value of BUN) -(0.008 x dialysis time)] + [4 -(3.5 x post-dialysis value of BUN/pre-dialysis value of BUN)] x (amount of drainage/post-dialysis body weight)}. Blood examinations Non-fasting blood

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samples were obtained while patients were lying in bed in a supine position after at least 15 minutes of rest on the 1st dialysis day of the week.The following pre-dialysis parameters were measured: hemoglobin (Hb), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride (TG), albumin-corrected calcium, inorganic phosphorus, intact parathyroid hormone, plasma renin activity, plasma aldosterone concentration, aldosterone to renin ratio, HbA1c, and creatinine (Cre), uric acid, C-reactive protein (CRP), and albumin (Alb) levels.Pre-dialysis serum s(P)RR levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Takara Bio Inc., Otsu City, Japan) consisting of a solid-phase sandwich ELISA with highly speci c antibodies for each protein. 24e following post-dialysis values were measured using conventional methods at an external testing laboratory (Kishimoto, Inc., Tomakomai City, Japan): human atrial natriuretic peptide (hANP), a marker of body uid volume 25,26,27 and BNP. Echocardiography Echocardiography was performed on a non-dialysis day, as previously described, 28 using the Vivid S6 System (GE Healthcare, Milwaukee, WI, USA), and cardiac functions were estimated as follows: 1) left ventricular ejection fraction, as a marker of contractile activity; 2) left ventricular mass index, as a marker of cardiac hypertrophy 29 ; and 3) E/e and deceleration time, as markers of left ventricular diastolic function. 30rotid intima-media thickness Ultrasonographic examinations of the common carotid artery, bulb, and internal carotid artery were bilaterally performed on a non-dialysis day, as described previously, 31 using the Nemio 30 Ultrasound System (Toshiba Medical Systems Co., Ltd, Tochigi, Japan). Ankle-brachial index and brachial-ankle pulse wave velocity The ankle-brachial index (ABI) (average and

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lower values) and brachial-ankle pulse wave velocity (baPWV) values (average and higher values) were measured on a non-dialysis day using a volume-plethysmographic apparatus PWV/ABI (Omron Healthcare Co., Ltd., Kyoto, Japan) following previously described methods. 32he BaPWV cannot be properly estimated when the ABI is lower than 0.9 because arterial occlusion retards the baPWV. 33,34Therefore, patients with ABI < 0.9 were excluded from the baPWV analysis. Computed tomography Body fat distribution was determined on a non-dialysis day using computed tomography (CT) imaging with a 64-row multislice CT scanner (Aquilion 64; Toshiba, Tokyo, Japan).The subcutaneous and visceral fat areas were measured at the level of the umbilicus using the Ziostation 2 software (Ziosoft, Tokyo, Japan). New-onset cardiovascular events or malignant diseases The occurrence of cardiovascular events or malignant diseases was investigated by either verifying with the electronic medical record for patients who had been undergoing dialysis at our a liated clinic for ve years or through telephone or letter.Cardiovascular events were de ned as 1) cardiovascular death, 2) nonfatal myocardial infarction, 3) unstable angina, 4) heart failure, 5) cerebral infarction or cerebral hemorrhage, and 6) severe lower limb ischemia (severe arteriosclerosis obliterans).Malignant diseases include stomach, colorectal, small intestinal, lung, renal, bladder, ureteral, penile, laryngeal, thyroid gland, and breast cancers, uterine neoplasms, and malignant melanomas. Study protocols The pre-dialysis serum s(P)RR levels on the 1st dialysis day of the week were measured at the start of the study.The patients were divided into two groups (higher and lower groups) according to the serum s(P)RR values, and the two groups were

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compared in terms of background factors and blood, physiological function, and CT data.Patients were followed up for 60 months or until death from any cause, and the relationship between serum s(P)RR levels and new-onset cardiovascular events or malignant diseases was investigated using annual physiological tests, such as abdominal echo and CT imaging.In addition, the association between serum s(P)RR values and all-cause, cardiovascular, and non-cardiovascular mortalities was investigated between the two groups. Statistical analyses Normally distributed continuous variables are expressed as means ± standard deviations and non-normally distributed ones as medians with interquartile ranges (25th and 75th percentiles).Intergroup comparisons of parameters were performed using the Wilcoxon signed-rank or Mann-Whitney U test.Categorical variables are presented as the number of patients and compared using the chi-square test.Kaplan-Meier plots and log-rank tests were also used to compare the all-cause, cardiovascular, and non-cardiovascular mortalities between the two groups (higher versus lower serum s(P)RR values).The C-index and area under the curve values were used to evaluate the effects of speci c variables on survival. 35Background factors contributing to cardiovascular mortality were analyzed using univariate Cox proportional hazard regression. In addition, we constructed multivariate Cox proportional hazard regression models to estimate the hazard ratios (HR) and 95% con dence intervals (95% CI) for cardiovascular mortality using factors showing signi cant correlations as covariates with mortality.The level of signi cance was set at P < 0.05.All analyses were performed using the Bell Curve for Excel (Social Survey Research Information Co. Ltd., Tokyo, Japan). Characteristics of the study patients at baseline All 255 patients, excluding

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three who received renal transplants during the study period, could be consecutively followed up for 60 months or until death [60 (38-60)].The median serum s(P)RR value of these patients at baseline was 29.8 ng/ml.Table 1 details the baseline characteristics of the study patients in the two groups (higher group, serum s(P)RR level ≥ 29.8 ng/ml; lower group, < 29.8 ng/ml) and includes background factors and blood, physiological function, and CT data.The number of patients with ABI measurements was 247 (96.9%).ABI was < 0.9 in 44 patients and as a result 203 patients underwent the baPWV analysis.When comparing between higher and lower groups of serum s(P)RR concentration, the CTR, Hb, TG, and CRP levels were signi cantly greater, and the highest and lowest SBP values and those at the beginning and end of the dialysis were signi cantly smaller in the higher group than in the lower group.The percentage of patients taking RAS-inhibitors (RAS-Is), such as angiotensin receptor blockers, angiotensin-converting enzyme inhibitors, and renin inhibitors, was signi cantly greater in the lower group than in the higher group (83.7% vs. 69.0%).Primary disease (presence or absence of diabetes mellitus) was not signi cantly different between the two groups.In addition, the BMI and Kt/V did not signi cantly differ between the two groups, suggesting that HD parameters may not strongly in uence the serum s(P)RR levels.There were 121 (47.4%) patients with a past history of cardiovascular events; 13 (4.7%) had nonfatal myocardial infarction, 41 (16.1%) unstable angina, 29 (11.4%)heart failure, 56 (22.0%) cerebral infarction or cerebral hemorrhage, and 14

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(5.5%) severe lower limb ischemia (severe arteriosclerosis obliterans).There were 27 (10.6%)patients with a past history of multiple cardiovascular events.Thirty-nine patients (15.1%) had a past history of malignant diseases; 10 (3.9%) had stomach cancer, eight (3.1%) colorectal cancer, one (0.4%) small intestinal cancer, four (1.6%) lung cancer, six (2.3%) renal cancer, four (1.6%) bladder cancer, one (0.4%) ureteral cancer, one (0.4%) penile cancer, one (0.4%) laryngeal cancer, one (0.4%) thyroid gland cancer, three (1.2%)breast cancer, one (0.4%) uterine neoplasm, and one (0.4%) malignant melanoma. There were two patients (0.8%) with a past history of multiple malignant diseases.The ratios of the past histories of cardiovascular events and malignant diseases were not signi cantly different between the groups. Estimating the risk of new-onset cardiovascular events During the 60-month follow-up period, 115 patients recorded new-onset cardiovascular events; 27 died of cardiovascular causes (23.5%), 5 had nonfatal myocardial infarction (4.3%), 26 unstable angina (22.6%), 12 heart failure (10.4%), 24 cerebral infarction or cerebral hemorrhage (20.9%), and 21 severe lower limb ischemia (severe arteriosclerosis obliterans) (18.3%).The serum s(P)RR levels were signi cantly higher in patients with new-onset cardiovascular events (31.2 ± 6.1 ng/ml, n = 115) than in those without them (29.8 ± 6.1 ng/ml, n = 140); P = 0.039.The serum s(P)RR levels in patients who developed severe lower limb ischemia were particularly high (Table 2).The incidence of new-onset cardiovascular events at the end of the follow-up period was signi cantly higher in patients with higher serum s(P)RR levels (52.8%) than in those with lower serum s(P)RR levels (38.3%);P = 0.010.The

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cumulative incidence of new-onset cardiovascular events was signi cantly greater in patients with higher serum s(P)RR concentrations than in those with lower s(P)RR concentrations (Figure 1). Estimating the risk of new-onset malignant diseases During the 60-month follow-up period, 38 patients recorded new-onset malignant diseases.At the end of the follow-up period, the serum s(P)RR levels did not signi cantly differ between patients with new-onset malignant diseases [30.5 (26.5-34.4)ng/ml, n = 38] and those without (30.4± 6.2 ng/ml, n = 217); P = 0.420.There was no difference in serum s(P)RR levels depending on the malignant-disease type (Table 3). There was no difference in the incidence of new-onset malignant diseases between patients with higher serum s(P)RR levels (16.5%) and those with lower levels (15.6%);P = 0.422. Association between serum s(P)RR levels and prognosis During the 60-month follow-up period, 106 deaths (41.6%) were recorded, including 63 cardiovascular deaths (24.7%)-2 due to acute myocardial infarction, 27 due to congestive heart failure, 15 due to lethal arrhythmia, and 6 due to cerebral hemorrhage, and 13 sudden unexpected deaths.Forty-three noncardiovascular deaths (16.9%) were recorded-15 due to infectious diseases, 21 due to cachexia, 5 due to cancer, and 2 due to liver failure.The 1-year, 2-year, 3-year, 4-year, and 5-year survival rates were 91.8%, 85.5%, 76.1%, 69.0%, and 58.0%, respectively.At the end of the follow-up period, the serum s(P)RR levels were signi cantly higher in patients who died by any cause (31.8 ± 5.8 ng/ml, n = 106) and in those who died of cardiovascular causes (32.5 ± 6.5 ng/ml, n = 63) than in

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those who survived (29.5 ± 6.3 ng/ml, n = 149 and 29.7 ± 5.8 ng/ml, n = 192, respectively; P = 0.005, <0.001, respectively).The higher serum s(P)RR group had higher rates of total (49.6%) and cardiovascular (33.9%) deaths than the lower serum s(P)RR group (total: 33.6% and cardiovascular: 15.6%, respectively) (P = 0.005 and P <0.001, respectively).Kaplan-Meier analyses showed that the cumulative survival rate of total death in the higher serum s(P)RR group was not signi cantly lower than that in the lower group (log-rank test, χ2 = 3.0, P = 0.083), but the cumulative survival rate of cardiovascular death in the higher group was signi cantly lower than that in the lower group (log-rank test, χ2 = 11.1,P < 0.001) (Figure 2).A receiver operating characteristic curve was constructed to determine the optimal cutoff value of the serum s(P)RR level for cardiovascular death (Figure 3).The optimal cutoff value of the serum s(P)RR level for cardiovascular death was 29.4 ng/ml (sensitivity, 0.778; speci city, 0.526) (Figure 3).Univariate Cox regression analyses showed that age, primary disease (diabetes mellitus) status; the difference (the highest -the lowest) in SBP values; medications (statin); CTR, s(P)RR, TG, hANP, BNP, and HbA1c values; max carotid intima-media thickness; and baPWV (average and higher values) were signi cantly and positively correlated while SBP (highest values), SBP (lowest values), SBP values at the end, medications (RAS-I), medications (calcium channel blockers), Cre, Alb, and ABI (average and lower values) were signi cantly and negatively correlated with cardiovascular death (Table 4).The results of the multivariate Cox regression analyses

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for cardiovascular mortality are shown in Table 5.All factors that were signi cantly correlated with total death in the univariate analyses (Table 4) were used as covariates in model 1.Factors that correlated with cardiovascular death, excluding ABI and baPWV, were used as covariates in model 2.Although the s(P)RR level was not signi cantly correlated with cardiovascular death in model 1, it showed a signi cant positive relationship with cardiovascular death in model 2 (HR: 1.041, 95% CI: 1.001-1.083,P = 0.046). Discussion The present study investigated the relationship between high serum s(P)RR levels and the occurrence of cardiovascular events or malignant diseases and the prognosis in patients undergoing HD; the study produced three major ndings.First, the CTR, Hb, TG, and CRP levels were signi cantly higher, and several SBP values were signi cantly lower in the higher serum s(P)RR group than in the lower serum s(P)RR group. Second, the occurrence ratio of cardiovascular events was signi cantly higher in the higher serum s(P)RR group than in the lower serum s(P)RR group.Finally, the serum s(P)RR level was independently associated with cardiovascular mortality, suggesting that increased expression of (P)RR may be associated with the progression of cardiovascular events in patients undergoing HD. In the present study, atherogenic factors, such as the CTR, TG, and CRP levels, were found to be greater in the higher serum s(P)RR group than in the lower serum s(P)RR group (Table 1) in accordance with the ndings of our previous report. 18 previously reported that the serum s(P)RR level was associated with atherosclerosis, independent of

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other risk factors, in patients undergoing HD, 18 and a high serum s(P)RR level was associated with an increase in BNP, independent of other risk factors. 20From a clinical perspective, these considerations suggest that patients with elevated s(P)RR concentrations should be screened for CVD risk factors. Conversely, there was no difference between the higher and lower s(P)RR groups with respect to their past histories of CVD or malignant diseases (Table 1).These results suggest that the serum s(P)RR concentration at the time of measurement may not be affected by the past history of cardiovascular events or malignant diseases. In this study, the serum s(P)RR levels were signi cantly higher in patients who experienced cardiovascular events during the follow-up period than in patients who did not; vice versa the incidence of cardiovascular events was signi cantly higher in patients with higher serum s(P)RR levels than in those with lower serum s(P)RR levels (Figure 1).We previously reported that high serum s(P)RR levels in patients undergoing HD were associated with severe atherosclerosis of the lower limbs, independent of other risk factors. 18herefore, we considered it is possible that serum s(P)RR could be used as a marker for atherosclerotic conditions.We also reported that a high serum s(P)RR level was associated with an increase in BNP during the rst 1-year follow-up period, independent of other risk factors, 20 suggesting that increased expression of (P)RR may be associated with the progression of heart failure in patients undergoing HD.Accordingly, higher s(P)RR levels may be associated with increased occurrence of cardiovascular events in patients undergoing

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HD, and serum s(P)RR could be potentially used as a predictive marker for cardiovascular events in these patients. (P)RR acts as an adaptor protein that co-locates with the Wnt receptor complex and contributes to the activation of Wnt/β-catenin signaling, independent of RAS. 7 The Wnt/β-catenin signaling pathway plays a pivotal role in numerous biological processes, such as in embryonic development, tissue homeostasis, and carcinogenesis. 8Overexpression of (P)RR has been observed in various malignant diseases, including pancreatic, 12,36 brain, 13,37 colorectal, 38,39 breast, 11 adrenal, 14 and endometrial 40 cancers.Overexpression of (P)RR may contribute to cancer initiation and progression via the Wnt/β-catenin, RAS, mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase-protein kinase/protein kinase B/mammalian target of rapamycin pathways, as well as to V-ATPase function in various cancers. 41atients undergoing HD are at a high risk for malignant diseases in the kidneys, bladder, thyroid, and other endocrine organs. 21,22Although the reason for the increased risk of malignant diseases in patients undergoing HD remains unclear, there are several possible explanations, including chronic in ammation, inhibition of the immune system, 42 poor nutritional status, reduced antioxidant capacity, accumulation of carcinogens, 43 and dialysis-related factors. 44In the present study, however, there was no difference in the incidence of malignant diseases between patients with higher and lower serum s(P)RR levels (Table 3).The possible reasons for this lack of association between s(P)RR concentration and cancer are insu cient sample size and limited follow-up.Besides, we cannot exclude the possibility of other confounding factors that affect the occurrence of malignant diseases in patients undergoing

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HD masking the effects of (P)RR.This issue should be addressed in further investigations. Chronic kidney disease (CKD) is associated with the risk of developing CVD, 45 and the risk of total death, cardiovascular death, and hospitalization due to CVDs is high in patients with CKD. 46In keeping with these data, the present study showed high all-cause mortality (41.6%) and high cardiovascular mortality (24.7%) in patients undergoing HD during the 60-month follow-up period.In patients undergoing HD, age, primary disease (diabetes mellitus) status, intradialytic BP change, 47 intradialytic hypotension, 48 serum Alb level, 49 and medication (RAS-I) 50 are, in general, associated with prognosis.In line with these reports, our study showed that age, primary disease (diabetes mellitus) status, and intradialytic BP change were signi cantly and positively correlated and the lowest intradialytic BP, medications (RAS-Is), and Alb level were signi cantly and negatively correlated with cardiovascular deaths (Table 4).Although the cumulative survival rate of cardiovascular death in the higher s(P)RR group was signi cantly lower than that in the lower s(P)RR group (Figure 2), the difference was not apparent until 20 months after the initiation of the study.The reason for this remains unclear; however, as the two groups had similar cardiac functions, carotid intima-media thicknesses, ABI and baPWV values, and histories of CVD at the start of this study, su cient time may have been required for the differences to manifest (Table 1). This study showed for the rst time that the s(P)RR level is associated with cardiovascular mortality in patients undergoing HD.Atherosclerosis and vascular calci cation have

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been shown to be risk factors for CVD in patients with CKD. 51,52(P)RR-mediated ERK signal transduction, independent of the generation of angiotensin II or the activation of its receptor, contributed to the development of vascular complications. 53,54The serum s(P)RR concentration was associated with arteriosclerosis 18 and worsening of heart failure. 20We also showed that the long-term administration of a (P)RR blocker attenuated the development of cardiac brosis and hypertrophy. 55Therefore, although the mechanism by which the blood s(P)RR concentration is associated with cardiovascular mortality remains unclear, we suppose that a high s(P)RR concentration could be associated with cardiovascular mortality via increased tissue (P)RR expression and atherosclerosis and/or heart failure and subsequent cardiovascular events.Further studies are required to test this assumption. Several limitations of the present study warrant mention.First, our sample size was relatively small.Second, the present data from patients undergoing HD may have been modulated by HD therapy because s(P)RR may have been dialyzed to some extent. 14Third, the mechanisms by which the serum s(P)RR level is associated with background factors remain unclear.Further studies are required to clarify the role of serum s(P)RR in patients undergoing HD. In conclusion, high serum s(P)RR level in patients undergoing HD was associated with various background factors and the occurrence of cardiovascular events.Furthermore, a high serum s(P)RR level was independently correlated with cardiovascular mortality.Therefore, s(P)RR could potentially be used as a marker for the occurrence of cardiovascular events and cardiovascular mortality and thus, could be useful in selecting patients requiring intensive care.Further studies are needed to determine whether reducing the

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serum s(P)RR level would improve patient prognosis in this population.Intergroup comparisons of parameters without correspondence were performed using the Mann-Whitney U test and those with correspondence were performed using the Wilcoxon signed-rank test.In addition, categorical variables have been presented as the number of patients and compared using the chi-square test. Table 1 . Comparison of characteristics of the study participants at baseline between the higher and lower serum s(P)RR concentration groups Higher group

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Reducing Bridge Pier Scour Using Gabion Mattresses Filled with Recycled and Alternative Materials : Scour is caused by the erosive action of flowing water, which causes materials from the bed and the banks of a river to be moved or unsettled. Hydraulic structures can be drastically impacted as a result of scour, which is why it is one of the most common causes of bridge failure around the world. With a predicted increase in climate conditions, the subsequent failure of hydraulic structures due to scour is likely to proliferate as the flooding of waterways is projected to rise. This study aims to determine the viability of introducing alternative materials to a scour countermeasure used in construction—gabion models—in a bid to improve the sustainability of a project whilst providing suitable scour mitigation measures. Existing literature was examined to comprehend the di ff erent scour countermeasures used, as well as the use of alternative materials that can be used as a scour countermeasure. A laboratory experiment was then carried out using a bridge pier embedded in a flume channel protected by gabion mattresses filled with alternative materials—stone, clothing and plastic—to analyse their e ff ectiveness. The results demonstrate that stone filled gabions are most e ff ective at reducing bridge pier scour. However, recycled clothing as a gabion fill could prove to be a viable alternative in construction projects, potentially leading to reduced construction costs and greater sustainability. However, more research on a greater scale is required to test this thesis. Introduction Various investigations have been undertaken which

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aim to determine the effectiveness of reducing bridge pier scour using different countermeasures. Yoon [1] specifically investigated the use of wire gabions (boxes filled with stones wrapped in wire mesh), containing uniform stone in a flume. By varying the depth, coverage and thickness to length ratio, Yoon determined that gabion mattresses were more cost effective than using riprap stones as a countermeasure. Similarly, Akib et al. [2] looked at reducing local bridge pier scour by combining geobags filled with alternative materials (using crushed concrete and oil palm shells) and collars, providing research into environmentally friendly solutions to scour countermeasures. Akib et al. [2] concluded that using a geobag and steel collar was 96% more effective than other countermeasures tested. Scour There are three main forms of scour that occur, and it is mainly driven by an increase in flow rate, either due to flooding or changes to the flow of the river. 1. Local scour is related to the presence of a hydraulic structure and occurs directly around the hydraulic structure; a bridge pier or abutment is a good example of this. Local scour can lead to . Bridge scour is a predominant issue around the world; in the USA between 1989 and 2000, 503 bridge failures were examined and it was found that flooding and scour contributed to 53% of all of these failures [3]. The study outlined that the average age of a bridge before failure was 52.5 years, revealing that almost half of the bridges examined were failing earlier than their anticipated design

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life. In New Zealand, an annual budget of NZD 36 million is allocated to resolve scour actions as a result of flooding [4]. More relevant to this research proposal, in the UK between 1846 and 2013, there have been a recorded 100 incidents of rail bridge/culvert failures related to flooding and scour, leading to a number of closures to railway lines and 15 deaths [5]. These findings highlight that there is a high expense to bridge failures as a result of scour, in terms of human life as well as financially. One example is the Schoharie Creek bridge collapse in 1987, as shown in Figure 1, which occurred because of local flooding, intensifying scour which weakened the bridge piers' foundations causing them to collapse, which resulted in 10 fatalities. Eng 2020, 1, FOR PEER REVIEW 2 sediment being removed from the bed and subsequent undermining of the bridge foundation leading to failure. 2. Contraction scour can also affect hydraulic structures and tends to be caused by a reduction in the width of the river channel (naturally or manmade). In times of flooding, excess water from the adjacent floodplain may be channelled into the bridge opening, increasing flow rate and leading to potential undermining of the bridge foundation. 3. General scour encompasses any types of scour that occur naturally, not in the presence of hydraulic structures. Over time, natural scour occurs as a result of erosion and degradation; therefore, non-cohesive deposits, such as gravels and sands in rivers, are more likely to experience scour. However, during floods,

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the process of natural scour is rapidly increased. Local scour can take place in clear-water conditions, where flow velocity upstream (µ ) is lower than the velocity threshold (µTC) of the material surrounding the bridge pier and before general movement of sediment from the riverbed has occurred. Scour will only occur if the velocity threshold is exceeded, due to an increase in flow, possibly caused by a blockage in the river or installation of flow-altering measures, such as groynes or vanes. If the velocity threshold is exceeded, then sediment is eroded and removed from the scour hole and is deposited downstream. This will continue until no more material is removeable from the scour hole. In live-bed conditions, scour can also occur where µ is greater than µTC; sediment is then removed from around the bridge pier creating a scour hole. As the flow velocity upstream is greater than the material velocity threshold, sediment is continuously transported in and around the scour hole from upstream to offset the removal of local material from around the bridge pier. The depth of local scour can therefore be written as µ µ . Bridge scour is a predominant issue around the world; in the USA between 1989 and 2000, 503 bridge failures were examined and it was found that flooding and scour contributed to 53% of all of these failures [3]. The study outlined that the average age of a bridge before failure was 52.5 years, revealing that almost half of the bridges examined were failing earlier than their anticipated

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design life. In New Zealand, an annual budget of NZD 36 million is allocated to resolve scour actions as a result of flooding [4]. More relevant to this research proposal, in the UK between 1846 and 2013, there have been a recorded 100 incidents of rail bridge/culvert failures related to flooding and scour, leading to a number of closures to railway lines and 15 deaths [5]. These findings highlight that there is a high expense to bridge failures as a result of scour, in terms of human life as well as financially. One example is the Schoharie Creek bridge collapse in 1987, as shown in Figure 1, which occurred because of local flooding, intensifying scour which weakened the bridge piers' foundations causing them to collapse, which resulted in 10 fatalities. Horseshoe and Wake Vortex When a bridge pier obstructs flow in a stream, a boundary layer separation occurs, where a point in contact with the bridge pier reverses in direction from the upstream bed in the approach flow, Horseshoe and Wake Vortex When a bridge pier obstructs flow in a stream, a boundary layer separation occurs, where a point in contact with the bridge pier reverses in direction from the upstream bed in the approach flow, resulting in a horseshoe vortex [7]. This leads to the creation of a scour hole as shown in Figure 2. Various studies have investigated the causes of bridge scour by studying horseshoe vortices, as this is believed to be the primary factor in causing bridge scour. Eng 2020, 1, FOR

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PEER REVIEW 3 resulting in a horseshoe vortex [7]. This leads to the creation of a scour hole as shown in Figure 2. Various studies have investigated the causes of bridge scour by studying horseshoe vortices, as this is believed to be the primary factor in causing bridge scour. A strong horseshoe vortex occurs in front of a bridge pier whereas a weaker wake-vortex system occurs in the rear of a cylindrical bridge pier downstream [9]. The mean size of a primary horseshoe vortex size is approximately 20% of the pier diameter [10]. From these investigations, it is evident that local scour countermeasures are required primarily upstream of the bridge pier, where the stronger horseshoe vortex forms, as well as downstream of the bridge pier where the weaker wake vortex system forms. Scour countermeasures can be installed downstream of a bridge pier, such as bed sills. Bed sills can reduce the scour depth around bridge piers by more than 80% in optimum conditions and when the distance from the bed sill to the bridge pier is smaller, the greater the reduction in depth [11]. Scour holes can occur downstream of bed sills where it was discovered that higher bed sills had a lower relative maximum scour depth compared to lower bed sills [12]; this was due to the build-up of sediment on the sills taking longer on higher bed sills. Whilst this information is insightful and provides a solid indication of the interaction between hydraulic structures and their relation to scour, it is notable that the

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experiment was not conducted using model bridge piers, therefore these findings may not be applicable. Flow-Altering Scour Countermeasures Local scour has been extensively investigated by various researchers who have focused on methods which aim to reduce scour. Haque et al. [13] and Chang and Karim [14] investigated the use of sacrificial piles and concluded that piles reduce the scour depth. Park et al. [15] explored further the effect of debris accumulation at sacrificial piles, finding that an increase in debris further reduced scour depth. Ghorbani and Kells [16] and Odgaard and Wang [17] investigated the use of Iowa vanes (also called deflector vanes) and found that at certain positioning and angle, the scour depth can be reduced drastically by up to 87.7%. It is worth noting that the scour hole which typically forms at the bridge pier is transferred to the corner of the Iowa vane, meaning that the Iowa vane then becomes more prone to failure, and could impact the stability of the bridge if it does so. Jahangirzadeh et al. [18] and Zarrati [19] experimented with the impact of collars on bridge piers and both confirmed that a larger collar (W/D, where W = width of collar and D = pier diameter) resulted in a larger reduction in scour depths. The shape of the collar applied is also a factor in scour reduction (in the former experiment, rectangular collars provided a further 8% reduction in scour). The placement of a collar under the bed of the sediment also leads to a reduction in scour,

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although [7]. (b) Horseshoe and wake vortices around cylindrical bridge pier [8]. A strong horseshoe vortex occurs in front of a bridge pier whereas a weaker wake-vortex system occurs in the rear of a cylindrical bridge pier downstream [9]. The mean size of a primary horseshoe vortex size is approximately 20% of the pier diameter [10]. From these investigations, it is evident that local scour countermeasures are required primarily upstream of the bridge pier, where the stronger horseshoe vortex forms, as well as downstream of the bridge pier where the weaker wake vortex system forms. Scour countermeasures can be installed downstream of a bridge pier, such as bed sills. Bed sills can reduce the scour depth around bridge piers by more than 80% in optimum conditions and when the distance from the bed sill to the bridge pier is smaller, the greater the reduction in depth [11]. Scour holes can occur downstream of bed sills where it was discovered that higher bed sills had a lower relative maximum scour depth compared to lower bed sills [12]; this was due to the build-up of sediment on the sills taking longer on higher bed sills. Whilst this information is insightful and provides a solid indication of the interaction between hydraulic structures and their relation to scour, it is notable that the experiment was not conducted using model bridge piers, therefore these findings may not be applicable. Flow-Altering Scour Countermeasures Local scour has been extensively investigated by various researchers who have focused on methods which aim to reduce scour.

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Haque et al. [13] and Chang and Karim [14] investigated the use of sacrificial piles and concluded that piles reduce the scour depth. Park et al. [15] explored further the effect of debris accumulation at sacrificial piles, finding that an increase in debris further reduced scour depth. Ghorbani and Kells [16] and Odgaard and Wang [17] investigated the use of Iowa vanes (also called deflector vanes) and found that at certain positioning and angle, the scour depth can be reduced drastically by up to 87.7%. It is worth noting that the scour hole which typically forms at the bridge pier is transferred to the corner of the Iowa vane, meaning that the Iowa vane then becomes more prone to failure, and could impact the stability of the bridge if it does so. Jahangirzadeh et al. [18] and Zarrati [19] experimented with the impact of collars on bridge piers and both confirmed that a larger collar (W/D, where W = width of collar and D = pier diameter) resulted in a larger reduction in scour depths. The shape of the collar applied is also a factor in scour reduction (in the former experiment, rectangular collars provided a further 8% reduction in scour). The placement of a collar under the bed of the sediment also leads to a reduction in scour, although the benefit is quite low as the collar becomes part of the scour hole as shown in Figure 3. Flow-altering scour countermeasures have been investigated extensively compared with other forms of scour countermeasures, however, the flow-altering

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countermeasures are more effective at reducing scour compared with bed armouring countermeasures. These experiments are all examples of flow-altering countermeasures; other types of this measure include tetrahedron frames, bed sills, groynes and spurs (the latter tend to be deployed to prevent contraction scour as opposed to local scour). Eng 2020, 1, FOR PEER REVIEW 4 the benefit is quite low as the collar becomes part of the scour hole as shown in Figure 3. Flow-altering scour countermeasures have been investigated extensively compared with other forms of scour countermeasures, however, the flow-altering countermeasures are more effective at reducing scour compared with bed armouring countermeasures. These experiments are all examples of flow-altering countermeasures; other types of this measure include tetrahedron frames, bed sills, groynes and spurs (the latter tend to be deployed to prevent contraction scour as opposed to local scour). Bed Armouring Scour Countermeasures Another type of scour countermeasure is bed armouring; countermeasures in this category include riprap protection, as shown in Figure 4, reno mattresses, gabion baskets, concrete slabs and armed soil with geotextile. Research has been undertaken in this area by Unger and Hager [21] and Lauchlan and Melville [22], where it was identified that the larger the size and area of the riprap placement, the greater the reduction of scour at bridge piers is. Although, it was found that when riprap stones fail and are transported, some may deposit in the scour hole, providing additional scour protection to the bridge pier [22], and therefore may provide skewed results. Akib et al. [24] investigated the

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use of geobags at skewed bridge piers, discovering that geobags filled with recycled crushed concrete is a highly effective method to reduce local bridge pier scour, with results ranging from 50% to 81%, all at varying flow velocities. Whilst the results showed that the larger the size of the concrete used in the geobag, the better the reduction in scour, it still showed that the geobags failed within 24 h, after being moved from their original location by the flow velocity. Bed Armouring Scour Countermeasures Another type of scour countermeasure is bed armouring; countermeasures in this category include riprap protection, as shown in Figure 4, reno mattresses, gabion baskets, concrete slabs and armed soil with geotextile. Research has been undertaken in this area by Unger and Hager [21] and Lauchlan and Melville [22], where it was identified that the larger the size and area of the riprap placement, the greater the reduction of scour at bridge piers is. Although, it was found that when riprap stones fail and are transported, some may deposit in the scour hole, providing additional scour protection to the bridge pier [22], and therefore may provide skewed results. Eng 2020, 1, FOR PEER REVIEW 4 the benefit is quite low as the collar becomes part of the scour hole as shown in Figure 3. Flow-altering scour countermeasures have been investigated extensively compared with other forms of scour countermeasures, however, the flow-altering countermeasures are more effective at reducing scour compared with bed armouring countermeasures. These experiments are all examples of flow-altering countermeasures; other

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types of this measure include tetrahedron frames, bed sills, groynes and spurs (the latter tend to be deployed to prevent contraction scour as opposed to local scour). Bed Armouring Scour Countermeasures Another type of scour countermeasure is bed armouring; countermeasures in this category include riprap protection, as shown in Figure 4, reno mattresses, gabion baskets, concrete slabs and armed soil with geotextile. Research has been undertaken in this area by Unger and Hager [21] and Lauchlan and Melville [22], where it was identified that the larger the size and area of the riprap placement, the greater the reduction of scour at bridge piers is. Although, it was found that when riprap stones fail and are transported, some may deposit in the scour hole, providing additional scour protection to the bridge pier [22], and therefore may provide skewed results. Akib et al. [24] investigated the use of geobags at skewed bridge piers, discovering that geobags filled with recycled crushed concrete is a highly effective method to reduce local bridge pier scour, with results ranging from 50% to 81%, all at varying flow velocities. Whilst the results showed that the larger the size of the concrete used in the geobag, the better the reduction in scour, it still showed that the geobags failed within 24 h, after being moved from their original location by the flow velocity. Akib et al. [24] investigated the use of geobags at skewed bridge piers, discovering that geobags filled with recycled crushed concrete is a highly effective method to reduce local bridge pier

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scour, with results ranging from 50% to 81%, all at varying flow velocities. Whilst the results showed that the larger the size of the concrete used in the geobag, the better the reduction in scour, it still showed that the geobags failed within 24 h, after being moved from their original location by the flow velocity. Similarly, Korkut et al. [25] found that at bridge abutments when collections of geobags were tested, they reduced scour depth; however, failure at the perimeter of the geobags occurs, leading to transportation from the flow into the scour hole. The failure of geobags and transportation into the scour hole could provide further scour protection to the bridge abutment, which is supported by Lauchlan and Melville's [22] research into the use of riprap stones. The investigation by Korkut et al. [25] also determined that geobags should be placed under the sediment depth to increase effectiveness against scour. It must be noted that both the geobag investigations feature different bridge foundations (skewed pier compared to abutments), therefore results could potentially fluctuate even if the bridge foundations are similar, such as a skewed pier and a regular pier. Limited research has been conducted in the use of cable-tied blocks (concrete blocks/slabs which are connected) as a scour countermeasure. Parker et al. [26] found that cable-tied blocks are prone to failure by uplift and, in absence of geotextile, leading to settlement of the cable-tied blocks due to leaching of sand from pores of the mattress. When a partial geotextile was used, results improved vastly,

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particularly due to the concrete blocks acting as an anchor to the geotextile. In some cases, the location of scour was transferred from the bridge pier to the edge of the cable-tied blocks; whilst scour was not eliminated, it had a favourable effect as it helped to bury the block further and anchor the mattress. Hoe [27] considered using cable-tied blocks in the form of ceramic tiles at a 5 mm thickness surrounding bridge abutments. Whilst significant amounts of scour developed downstream from the bridge abutment, the spill-through slope downstream of the abutment did not fail. The implementation of a geotextile could have contributed to a reduction in scour; however, it must be noted that the experiment was conducted at bridge abutments, therefore geotextile may not have been as effective as at bridge piers. Cable-tied blocks with geotextile should be considered as a viable alternative to riprap in sand bed streams. Pier Adjustment Scour Countermeasures More recently, there have been investigations into newer methods of local scour countermeasures which focus on changing the properties of the bridge pier by introducing a bridge pier slot or by altering the pier shape and inclination. Karimi et al. [28] and Kitsikoudis et al. [29] studied the effect of pier inclination on bridge pier scour and both supported each other's findings that higher inclination angles led to a greater reduction in scour depth compared to other inclination angles tested during their relative experiments. The reduction in scour is due to the reduction in the wake vortex depth and area coverage.

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Varying the bridge pier shape can also be effective in reducing scour; Murtaza et al. [30] investigated the use of square, circular, oval and octagonal pier shapes. Findings from this research discovered that octagonal pier shapes resulted in the largest scour reduction and square pier shapes were the worst performing in reducing scour. Oval and circular pier shapes provided mostly results within 10% of each other, whilst all pier shapes displayed evidence of scouring in the upstream side of the pier. Likewise, Farooq and Ghunman [31] found similar results, determining that square-shaped piers resulted in the greatest scour depth results, whereas the octagonal-shaped pier led to the greatest percentage scour reduction (34% when pier size was 5 cm and 31% when bed material median grain size was increased). Furthermore, the research revealed that scour profiles were similar in that the upstream face of the pier had a larger scour depth compared to the downstream face. Hajikandi and Golnabi [32] investigated changing the various configuration of slots on bridge piers, including Y, T and straight slots. Results revealed that the installation of any slot reduces the scour dimensions, depth and width; straight slots were found to be the most effective out of the three Eng 2020, 1 193 slot types tested, resulting in a 38% scour reduction compared to 33% from Y-shaped slots. The depth of the embedment of the slot towards the bed material also led to reduction of the scour depth in all slot types. An example of a pier slot is shown in Figure

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5. Eng 2020, 1, FOR PEER REVIEW 6 Figure 5. Diagram of rectangular pier slot combined with a downstream bed sill used to reduce bridge scour [11]. Obied and Khassaf [33] tested the impact of rectangular slots with rounded edges on scour depth, and the results identified that increasing the slot length leads to a proportional decrease in the scour depth ratio. Through testing five different slot lengths, scour depth reduction increased from 31% to 49% as slot length increased. Similar to the findings of Hajikandi and Golnabi [32], this research highlights that adding a slot to a bridge pier is an effective method to reduce scour, due to the downstream flow being diverted and the horseshoe vortex being reduced, and it also suggests that additional measures including embedment and slot length may also provide further reductions. Whilst installing slots to a bridge pier reduces scour, it poses the risk of also reducing the structural strength of the bridge pier due to the loads from the bridge not having a suitable pathway to transfer to the bridge foundations. A weakened bridge pier could lead to collapse. To prevent this, the bridge foundations may require larger or stronger foundations to compensate for the loss of strength, therefore making the insertion of a slot into the bridge pier potentially less viable. Gabion Scour Countermeasures Gabions are wire mesh baskets filled with stones of varying size as shown in Figure 6. There are various types of gabions, including sack gabions, gabion mattresses and box gabions, which vary in shape,

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size and use. Little research has been performed into the use of gabions for reducing bridge pier scour, and current practice involves using gabions as revetment along river channels. Gabions are at risk of failure in several ways; they may pull away from the bridge pier if exposed to excessive edge scour, leading to the failure of the gabion, typically caused by winnowing, which is the removal of fine material (of the sediment), dependant on the size of particles of two different sediments. The wire used in the gabion is however the most likely to fail; it can be damaged from corrosion (if the water contains contaminants), abrasion from debris accumulation or if excessive settlement occurs it could lead to failure of the wire in tension as the gabion deforms; therefore, lacing wire should be used to tie the gabion mattresses together. All these failure mechanisms would likely lead to the transport of fill material downstream, and therefore the placement of gabions into certain waterways may not be viable; for example, coarse bed rivers. Yoon [1] investigated the use of wire gabions filled with stone, finding that as the length to thickness ratio increased, the gabion stability also increased, up to a limiting factor of L/t = 3. Any value greater than L/t = 3 is likely to induce extra material costs which may offset any additional gabion stability obtained, demonstrating that increasing L/t is not advantageous. Furthermore, wire gabions can provide better performance in reducing scour than ripraps of the same size, or equivalent sizing

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of gabions can result in more cost-effective solutions to reducing scour [1]. However, the gabion sizing equation used in this research is based on riprap sizing equations found in Lauchlan Obied and Khassaf [33] tested the impact of rectangular slots with rounded edges on scour depth, and the results identified that increasing the slot length leads to a proportional decrease in the scour depth ratio. Through testing five different slot lengths, scour depth reduction increased from 31% to 49% as slot length increased. Similar to the findings of Hajikandi and Golnabi [32], this research highlights that adding a slot to a bridge pier is an effective method to reduce scour, due to the downstream flow being diverted and the horseshoe vortex being reduced, and it also suggests that additional measures including embedment and slot length may also provide further reductions. Whilst installing slots to a bridge pier reduces scour, it poses the risk of also reducing the structural strength of the bridge pier due to the loads from the bridge not having a suitable pathway to transfer to the bridge foundations. A weakened bridge pier could lead to collapse. To prevent this, the bridge foundations may require larger or stronger foundations to compensate for the loss of strength, therefore making the insertion of a slot into the bridge pier potentially less viable. Gabion Scour Countermeasures Gabions are wire mesh baskets filled with stones of varying size as shown in Figure 6. There are various types of gabions, including sack gabions, gabion mattresses and box gabions, which

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vary in shape, size and use. Little research has been performed into the use of gabions for reducing bridge pier scour, and current practice involves using gabions as revetment along river channels. Gabions are at risk of failure in several ways; they may pull away from the bridge pier if exposed to excessive edge scour, leading to the failure of the gabion, typically caused by winnowing, which is the removal of fine material (of the sediment), dependant on the size of particles of two different sediments. The wire used in the gabion is however the most likely to fail; it can be damaged from corrosion (if the water contains contaminants), abrasion from debris accumulation or if excessive settlement occurs it could lead to failure of the wire in tension as the gabion deforms; therefore, lacing wire should be used to tie the gabion mattresses together. All these failure mechanisms would likely lead to the transport of fill material downstream, and therefore the placement of gabions into certain waterways may not be viable; for example, coarse bed rivers. and Melville [22]; therefore, due to the lack of research into gabion models, it may not provide an accurate sizing method for gabion baskets. Figure 6. Example of a stepped gabion mattress formation to prevent bridge pier scour [34]. Lagasse et al. [35] concludes that the best performance of gabion mattresses as a pier scour countermeasure occurs when mattresses are extended a distance at least double the pier width in all directions. A geotextile filter also provides further stability

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to the mattress, similar to Parker et al.'s findings [26]. Higher debris loads can be detrimental to gabion mattresses [35], which is validated by Pagliara et al. [36], which suggests that debris accumulation may reduce the efficiency of gabions. Although there is a current practice of using gabions in construction, there is a lack of research on the effect of gabions on reducing bridge pier scour. With the increase in global warming expected to reach 1.5 °C between 2030 and 2052 [37], severe weather developments could be a consequence. In the future, severe rainfall in both summer and winter could lead to flash flooding in the UK [38], and this in turn could lead to scouring at bridge piers, resulting in high financial costs and fatalities, not only during the flooding peaks but also as flow recedes [39]. The introduction of recyclable materials into gabions could provide a solution to scouring at bridge piers, as well as contributing to sustainable and cost-effective construction by reusing material compared to alternatives such as riprap, which requires twice the rock size as the ones used in gabion mattresses to provide the same stability [26]. Materials and Methods As previously discussed, data produced from the investigation of gabion baskets has been relatively limited with the most recent dataset being produced by Yoon [1]. Whilst Lagasse et al. [22] partly investigated the effect of gabion mattresses in reducing bridge pier scour, both sources are outdated to have been used for datasets. No research has been conducted into the use of gabion

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baskets constructed with recyclable materials. Therefore, primary data was collected by performing an experiment in order to obtain depth of the scour by measuring from the pier to the bottom of the scour hole. This is required as it is needed to calculate the scour hole depth and subsequent scour reduction percentage when compared with values from the different variables tested. Design Methodology The design methodology was mainly performed by changing variables throughout the experiment including: the type of material used in the gabion baskets, placement depth of the gabion baskets, thickness of gabion baskets and depth of flow used in the flume. By using grade 304 stainless steel wire mesh with an aperture of 1.2 mm, it was then possible to model the gabions. The wire mesh Yoon [1] investigated the use of wire gabions filled with stone, finding that as the length to thickness ratio increased, the gabion stability also increased, up to a limiting factor of L/t = 3. Any value greater than L/t = 3 is likely to induce extra material costs which may offset any additional gabion stability obtained, demonstrating that increasing L/t is not advantageous. Furthermore, wire gabions can provide better performance in reducing scour than ripraps of the same size, or equivalent sizing of gabions can result in more cost-effective solutions to reducing scour [1]. However, the gabion sizing equation used in this research is based on riprap sizing equations found in Lauchlan and Melville [22]; therefore, due to the lack of research into gabion models, it may not

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provide an accurate sizing method for gabion baskets. Lagasse et al. [35] concludes that the best performance of gabion mattresses as a pier scour countermeasure occurs when mattresses are extended a distance at least double the pier width in all directions. A geotextile filter also provides further stability to the mattress, similar to Parker et al.'s findings [26]. Higher debris loads can be detrimental to gabion mattresses [35], which is validated by Pagliara et al. [36], which suggests that debris accumulation may reduce the efficiency of gabions. Although there is a current practice of using gabions in construction, there is a lack of research on the effect of gabions on reducing bridge pier scour. With the increase in global warming expected to reach 1.5 • C between 2030 and 2052 [37], severe weather developments could be a consequence. In the future, severe rainfall in both summer and winter could lead to flash flooding in the UK [38], and this in turn could lead to scouring at bridge piers, resulting in high financial costs and fatalities, not only during the flooding peaks but also as flow recedes [39]. The introduction of recyclable materials into gabions could provide a solution to scouring at bridge piers, as well as contributing to sustainable and cost-effective construction by reusing material compared to alternatives such as riprap, which requires twice the rock size as the ones used in gabion mattresses to provide the same stability [26]. Materials and Methods As previously discussed, data produced from the investigation of gabion baskets has been

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relatively limited with the most recent dataset being produced by Yoon [1]. Whilst Lagasse et al. [22] partly investigated the effect of gabion mattresses in reducing bridge pier scour, both sources are outdated to have been used for datasets. No research has been conducted into the use of gabion baskets constructed with recyclable materials. Therefore, primary data was collected by performing an experiment in order to obtain depth of the scour by measuring from the pier to the bottom of the scour hole. This is required as it is needed to calculate the scour hole depth and subsequent scour reduction percentage when compared with values from the different variables tested. Design Methodology The design methodology was mainly performed by changing variables throughout the experiment including: the type of material used in the gabion baskets, placement depth of the gabion baskets, thickness of gabion baskets and depth of flow used in the flume. By using grade 304 stainless steel wire mesh with an aperture of 1.2 mm, it was then possible to model the gabions. The wire mesh was cut into varying dimensions and filled with different types of material, as shown in Figure 7, and the wire mesh was then bonded using epoxy resin. Eng 2020, 1, FOR PEER REVIEW 8 was cut into varying dimensions and filled with different types of material, as shown in Figure 7, and the wire mesh was then bonded using epoxy resin. Table 1 summarises the dimensions of the gabion models used. The materials used in models C8, C12 and

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C16 consisted of cotton recycled clothing cut into small squares of 5 mm × 5 mm × 1 mm. Likewise, the plastic used in P8, P12 and P16 was also cut into small squares of 5 mm × 5 mm × 1 mm. Due to nature of the stone aggregate used in models S8, S12 and S16, this material varied in size; however, the average size of aggregate was 5 mm. An acrylic circular straight rod with the dimensions 8 mm × 25 mm was selected to be used as the bridge pier model. Initially, the experiment analysed in this project was undertaken at Nottingham Trent University in the Hydraulics Laboratory in a Perspex flume channel of 5 m length, 0.08 m width and 0.25 m height, as prior research had established that the pier diameter should not be more than 10% of channel width [40] so that the sides of the Perspex channel did not interfere with the experiment. Table 1. Summary of gabion model dimensions. Material Type Gabion Length (mm) Gabion Thickness (mm) Model Label Stone 32 Raudkivi and Ettema [41] determined that if D/d50 (pier diameter/sediment size) is greater than 20-25, then the sediment size has a negligible impact on the scour hole; therefore, uniform sand sediment of a mean particle size (d50) 0.6 mm was used. In the experiment conducted, D/d50 = 8 mm/0.6 mm = 13.3 and may therefore impede the erosion process caused by the horseshoe and wake vortices. Raudkivi and Ettema [41] further clarified the impact of the value

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of D/d50, which in this research Table 1 summarises the dimensions of the gabion models used. The materials used in models C8, C12 and C16 consisted of cotton recycled clothing cut into small squares of 5 mm × 5 mm × 1 mm. Likewise, the plastic used in P8, P12 and P16 was also cut into small squares of 5 mm × 5 mm × 1 mm. Due to nature of the stone aggregate used in models S8, S12 and S16, this material varied in size; however, the average size of aggregate was 5 mm. An acrylic circular straight rod with the dimensions 8 mm × 25 mm was selected to be used as the bridge pier model. Initially, the experiment analysed in this project was undertaken at Nottingham Trent University in the Hydraulics Laboratory in a Perspex flume channel of 5 m length, 0.08 m width and 0.25 m height, as prior research had established that the pier diameter should not be more than 10% of channel width [40] so that the sides of the Perspex channel did not interfere with the experiment. Raudkivi and Ettema [41] determined that if D/d 50 (pier diameter/sediment size) is greater than 20-25, then the sediment size has a negligible impact on the scour hole; therefore, uniform sand sediment of a mean particle size (d 50 ) 0.6 mm was used. In the experiment conducted, D/d 50 = 8 mm/0.6 mm = 13.3 and may therefore impede the erosion process caused by the horseshoe and wake vortices. Raudkivi and

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Ettema [41] further clarified the impact of the value of D/d 50 , which in this research project will lead to a large proportion of energy dissipation of the downflow into material at the bottom of the scour hole. The coverage of the gabions was selected as the optimum performance of gabion mattresses, which occurred when mattresses extended 2 times the pier width in all directions around the pier [35]. Therefore, as the pier diameter was 8 mm, the gabions used in the experiment had dimensions of 32 mm width × 32 mm length in order to satisfy this constraint. The sketch of the experiment layout can be understood by Figure 8 shown below. Eng 2020, 1, FOR PEER REVIEW 9 had dimensions of 32 mm width × 32 mm length in order to satisfy this constraint. The sketch of the experiment layout can be understood by Figure 8 shown below. Testing Methodology The initial experiment conducted in the 5 m flume channel proved to be unsuccessful; the flow rate of the flume could not be regulated at a low enough rate to allow enough time to measure the scour hole depth without a substantial amount of sand sediment being transported downstream. Therefore, a smaller flume channel, as shown in Figure 8, was used, which was 2.5 m long and 0.05 m width with a height of 0.25 m. The pier diameter (8 mm) remained the same and therefore would not meet the recommendations of previous research in regard to the 10% of channel width [40].

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Although, alternative findings state that if the flume width to pier width is greater than 6.25, then the flume walls will have a negligible impact on the scour effect [41]. In the experiment conducted, the flume width to pier width ratio was calculated as 50 mm/8 mm = 6.25, which suggests that, theoretically, the flume walls should have no impact on the scour development around the bridge pier. The bridge pier was installed at a distance 1.25 m from the start of the flume surrounded by the gabion model, with the sand sediment installed at a depth of 40 mm or 50 mm (depending on sediment depth selected) extending 0.25 m either side of the bridge pier, and then gradually sloped to 0 mm of the bed, as shown in Figure 9. The flume was filled slowly with water until the sand sediment was submerged. Once completed, the depth from the top of the pier was recorded to the top of sand sediment, as well as from the top of the pier to the bottom of the scour hole, which allowed for the calculation of the scour hole depth per gabion model at different time intervals. The flow rate, Q, was maintained at a constant rate of 0.0001 m 3 /s and was monitored using a Testing Methodology The initial experiment conducted in the 5 m flume channel proved to be unsuccessful; the flow rate of the flume could not be regulated at a low enough rate to allow enough time to measure the scour hole

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depth without a substantial amount of sand sediment being transported downstream. Therefore, a smaller flume channel, as shown in Figure 8, was used, which was 2.5 m long and 0.05 m width with a height of 0.25 m. The pier diameter (8 mm) remained the same and therefore would not meet the recommendations of previous research in regard to the 10% of channel width [40]. Although, alternative findings state that if the flume width to pier width is greater than 6.25, then the flume walls will have a negligible impact on the scour effect [41]. In the experiment conducted, the flume width to pier width ratio was calculated as 50 mm/8 mm = 6.25, which suggests that, theoretically, the flume walls should have no impact on the scour development around the bridge pier. The bridge pier was installed at a distance 1.25 m from the start of the flume surrounded by the gabion model, with the sand sediment installed at a depth of 40 mm or 50 mm (depending on sediment depth selected) extending 0.25 m either side of the bridge pier, and then gradually sloped to 0 mm of the bed, as shown in Figure 9. The flume was filled slowly with water until the sand sediment was submerged. Once completed, the depth from the top of the pier was recorded to the top of sand sediment, as well as from the top of the pier to the bottom of the scour hole, which allowed for the calculation of the scour hole depth per gabion

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model at different time intervals. The flow rate, Q, was maintained at a constant rate of 0.0001 m 3 /s and was monitored using a digital hydraulic bench. A vertical sluice gate was installed at the end of the flume in order to control flow depth (Y1), which throughout the experiment was lowered at varied time intervals shown in Table 2. Measurements of Y1 and Y2 were not observed during the experiment, and therefore it was not possible to calculate the approach flow velocity and velocity threshold of the sediment. The inflow discharge of the sediment (Q s ) was also not considered during this experiment. gabion model, with the sand sediment installed at a depth of 40 mm or 50 mm (depending on sediment depth selected) extending 0.25 m either side of the bridge pier, and then gradually sloped to 0 mm of the bed, as shown in Figure 9. The flume was filled slowly with water until the sand sediment was submerged. Once completed, the depth from the top of the pier was recorded to the top of sand sediment, as well as from the top of the pier to the bottom of the scour hole, which allowed for the calculation of the scour hole depth per gabion model at different time intervals. The flow rate, Q, was maintained at a constant rate of 0.0001 m 3 /s and was monitored using a digital hydraulic bench. A vertical sluice gate was installed at the end of the flume in order to control flow depth

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(Y1), which throughout the experiment was lowered at varied time intervals shown in Table 2. Measurements of Y1 and Y2 were not observed during the experiment, and therefore it was not possible to calculate the approach flow velocity and velocity threshold of the sediment. The inflow discharge of the sediment (Qs) was also not considered during this experiment. Hypothesis It is anticipated that the research conducted in this project will contribute to the development of alternative materials in effectively reducing bridge pier scour. There is vast potential for this topic to become cost effective and sustainable whilst also being fully operational. It is predicted that the varying parameters used in the testing will directly contribute to the reduction or enhancement of a scour hole around the bridge pier; furthermore, it is predicted the test parameters may change the width and length of the scour hole. It is expected that the stone filled gabion models will be more effective at reducing scour hole depth than any other form of gabion models due to the aggregates' larger surface area compared to the smaller surface areas of clothing and plastic materials used in the other gabion models. Results The experiment was conducted first with the use of a control variable; initially, no gabions were used to surround the bridge pier at different sediment depths as this enabled a baseline of data to be generated so that later in the investigation comparable data could be calculated against it to produce a percentage in scour reduction. This is summarized in Table

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3. Effect of Gabion Fill-8 mm Thickness Firstly, gabions of 8 mm thickness consisting of different materials as shown in Table 4 were tested. The reduction in scour has been calculated from the original scour depths provided in Table 3 and have been summarised below in Table 4 and Figure 10. Figure 10. Effect of gabion fill type at 8 mm thickness on reducing total scour depth. Effect of Gabion Fill-12 mm Thickness The next gabions to be tested were a thickness of 12 mm, and the contents of the gabions remained unchanged (same materials as shown in Table 1). Results from the experiment are provided below in Table 5 and Figure 11. Table 5. Results of 12 mm thickness gabion models in reducing total scour depth. Effect of Gabion Fill-12 mm Thickness The next gabions to be tested were a thickness of 12 mm, and the contents of the gabions remained unchanged (same materials as shown in Table 1). Results from the experiment are provided below in Table 5 and Figure 11. Effect of Gabion Fill-12 mm Thickness The next gabions to be tested were a thickness of 12 mm, and the contents of the gabions remained unchanged (same materials as shown in Table 1). Results from the experiment are provided below in Table 5 and Figure 11. Table 5. Results of 12 mm thickness gabion models in reducing total scour depth. Effect of Gabion Fill-16 mm Thickness The final gabions to be tested were a thickness of 16 mm, with the contents of the

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