bluuebunny/crossref_metadata_2025_split huggingface dataset:
ПОЛУЧЕНИЕ В КЛЕТКАХ БАКТЕРИЙ ESCHERICHIA COLI РЕКОМБИНАНТНОГО ЧЕЛОВЕЧЕСКОГО БЕЛКА Р62
Authors: Сауткина Н.В. , Матчан И.Д. , Пластинина О.В. , Прокулевич В.А. 
Published: September 2024

Протеостаз включает внутриклеточные биологические пути, контролирующие биогенез, фолдинг, перенос и деградацию белков, присутствующих внутри и вне клетки. Эффективный протеостаз имеет решающее значение для поддержания соматического гомеостаза, а его снижение во время старения приводит к клеточной дисфункции и заболеваниям. Селективная аутофагия представляет собой форму аутофагии, опосредованную рецепторами, которые нацелены на специфические субстраты для деградации, и является важным процессом для поддержания протеостаза.
Белок секвестосома-1 (p62/SQSTM1) известен как убиквитин-связывающий белок p62 с доменной организацией. Он является классическим селективным рецептором аутофагии, но также выполняет определенные функции в убиквитин-протеасомной системе, клеточном метаболизме, передаче сигналов и апоптозе. Белок p62 играет решающую роль во множестве клеточных процессов, включая реакцию на повреждение ДНК, старение, инфекцию и иммунитет, хроническое воспаление и канцерогенез, зависящий от аутофагии или независимый от нее. Потеря p62 приводит к ускоренному старению клеток из-за снижения протеостаза, нарушения регуляции сигнальных путей и неспособности реагировать на окислительный стресс.
Целью работы является клонирование и экспрессия рекомбинантного человеческого белка p62 в клетках бактерий E. coli.
В процессе разработки дизайна гена, обозначенного как p62, определяли нуклеотидную последовательность путем обратной трансляции соответствующей первичной структуры белка p62 из базы данных UniProt, затем оптимизировали для усиления экспрессии в клетках бактерий E. coli до значения CAI=0,8. На 5’-конце фрагмента вводили сайт узнавания эндонуклеазы NdeI, на 3’-конце – сайт узнавания эндонуклеазы EcoRI и стоп-кодоны.
Ген p62 по соответствующим сайтам рестрикции вводили в состав вектора рЕТ-24b(+) (Novagen) под контролем промотора бактериофага Т7 и клонировали в клетках штамма E. coli XL1-Blue. Результаты клонирования проверяли ПЦР-анализом трансформантов с использованием праймеров, фланкирующих полилинкерную область вектора, а также рестрикционным анализом рекомбинантных плазмид.
Индуцировали экспрессию гена p62 в клетках штамма E. coli BL21-Gold(DEЗ) синтетическим аналогом лактозы – ИПТГ (0,5 ммоль/л) при 37 °С в течение 4 часов. Эффективность экспрессии анализировали при помощи SDS-PAGE электрофореза, результаты которого показали, что целевой белок массой около 48 кДа накапливается в бактериальных клетках.
Таким образом, получен бактериальный продуцент, в клетках которого рекомбинантный многофункциональный человеческий белок p62 синтезируется в количестве около 25 % от общего клеточного белка при стандартных условиях индукции экспрессии, рекомендованных производителями плазмид серии рЕТ.
3D Study of Cloud Cavitation on a Circular Leading-Edge Hydrofoil Using RANS Approaches
Authors: Puga Gallegos Diana , Luo Xianwu 
Published: September 2024

Abstract
In spite of the improvements in computational fluid dynamics (CFD) techniques, engineering problems still need robust models that can ensure accuracy at low cost in the prediction of complex phenomena such as cavitating turbulent flows. In the present study, cavitation over a circular leading-edge hydrofoil imitating the leading-edge geometry of centrifugal pump impeller blades was analyzed for different cavitation numbers ranging from 2 to 2.7 and Reynolds numbers of 1.3 × 106 and 1.6 × 106. The unsteady simulations were conducted by using the RANS method with the Zwart-Gerber-Belamri (ZGB) cavitation model. To capture the features of cavitating turbulent flow, three turbulence models i.e. the MSST PANS, k-ε FBDCM and k-ω FBDCM were evaluated to determine the suitable turbulence model for simulating cloud cavitation over a hydrofoil. The numerical results were compared with the experimental data obtained by Bohm and the sequence of images recorded by Dular. The results showed that the MSST PANS can reproduce better the cavitation phenomenon than the k-ε FBDCM and k-ω FBDCM turbulence models when the shedding frequency and the cavitating structures were analyzed and compared with the experiment.
Prospectively predicting BPaMZ phase IIb/III trial outcomes using a translational mouse-to-human platform
Authors: Goh Janice J. N. , Wang Qianwen , Zhang Nan , de Castro Suarez Niurys , Bustion Annamarie E. , Nuermberger Eric L. , Savic Rada 
Published: September 2024

ABSTRACT

Despite known treatments, tuberculosis (TB) remains the world’s top infectious killer, highlighting the pressing need for new drug regimens. To prioritize the most efficacious drugs for clinical testing, we previously developed a PK-PD translational platform with bacterial dynamics that reliably predicted short-term monotherapy outcomes in Phase IIa trials from preclinical mouse studies. In this study, we extended our platform to include PK-PD models that account for drug-drug interactions in combination regimens and bacterial regrowth in our bacterial dynamics model to predict cure at the end of treatment and relapse 6 months post-treatment. The Phase III STAND trial testing a new regimen comprised of pretomanid (Pa), moxifloxacin (M), and pyrazinamide (Z) (PaMZ) was suspended after a separate ongoing trial (NC-005) suggested that adding bedaquiline (B) to the PaMZ regimen would improve efficacy. To forecast if the addition of B would, indeed, benefit the PaMZ regimen, we applied an extended translational platform to both regimens. We predicted currently available short- and long-term clinical data well for drug combinations related to BPaMZ. We predicted the addition of B to PaMZ to shorten treatment duration by 2 months and to have similar bacteriological success to standard HRZE treatment (considering only treatment success but not withdrawal from side effects and other adverse events), both at the end of treatment for treatment efficacy and 6 months after treatment has ended in relapse prevention. Using BPaMZ as a case study, we have demonstrated our translational platform can predict Phase II and III outcomes prior to actual trials, allowing us to better prioritize the regimens most likely to succeed.
Phospholipid biosynthesis regulation for improving pigment production by
Monascus
in response to ammonium chloride stress
Authors: Jiang Xiaofei , Hong Xiya , Wang Zhulin , Liu Jun , Zhong Haiyan , Ren Jiali , Zhou Bo 
Published: September 2024

ABSTRACT

In the actual industrial production process, the efficient biosynthesis and secretion of
Monascus
pigments (MPs) tend to take place under abiotic stresses, which often result in an imbalance of cell homeostasis. The present study aimed to thoroughly describe the changes in lipid profiles in
Monascus purpureus
by absolute quantitative lipidomics and tandem mass tag-based quantitative proteomics. The results showed that ammonium chloride stress (15 g/L) increased MP production while inhibiting ergosterol biosynthesis, leading to an imbalance in membrane lipid homeostasis in
Monascus
. In response to the imbalance of lipid homeostasis, the regulation mechanism of phospholipids in
Monascus
was implemented, including the inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of the biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway rather than the Kennedy pathway. The inhibition of lysophospholipid biosynthesis was attributed to the upregulated expression of protein and its gene related to lysophospholipase NTE1, while maintenance of the PC/PE ratio was achieved by the upregulated expression of protein and its gene related to CTP: phosphoethanolamine cytidylyltransferase and phosphatidylethanolamine N-methyltransferase in the Kennedy pathway. These findings provide insights into the regulation mechanism of MP biosynthesis from new perspectives.

IMPORTANCE

Monascus
is important in food microbiology as it produces natural colorants known as
Monascus
pigments (MPs). The industrial production of MPs has been achieved by liquid fermentation, in which the nitrogen source (especially ammonium chloride) is a key nutritional parameter. Previous studies have investigated the regulatory mechanisms of substance and energy metabolism, as well as the cross-protective mechanisms in
Monascus
in response to ammonium chloride stress. Our research in this work demonstrated that ammonium chloride stress also caused an imbalance of membrane lipid homeostasis in
Monascus
due to the inhibition of ergosterol biosynthesis. We found that the regulation mechanism of phospholipids in
Monascus
was implemented, including inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway. These findings further refine the regulatory mechanisms of MP production and secretion.
Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2
Authors: Palmer Marley , Leo Alessandro , Atyeo Natalie , Tomacari Christiana , Nguyen Xuan , Papp Bernadett 
Published: September 2024

ABSTRACT

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.

IMPORTANCE
The dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.
Sodium-dependent phosphate transporter PiT1/SLC20A1 as the receptor for the endogenous retroviral envelope syncytin-B involved in mouse placenta formation
Authors: Mousseau Guillaume , Préault Noémie , Souquere Sylvie , Bireau Caroline , Cassonnet Patricia , Bacquin Agathe , Beck Laurent , Pierron Gérard , Jacob Yves , Dupressoir Anne , Heidmann Thierry 
Published: September 2024

ABSTRACT

Syncytins
are envelope genes of retroviral origin that play a critical role in the formation of a syncytial structure at the fetomaternal interface
via
their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one observed in all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryonic development. The cellular receptor for SynA has been identified as the membrane protein LY6E and is not the receptor for SynB. Here, by combining a cell–cell fusion assay with the screening of a human ORFeome-derived expression library, we identified the transmembrane multipass sodium-dependent phosphate transporter 1 PiT1/SLC20A1 as the receptor for SynB. Transfection of cells with the cloned receptor, but not the closely related PiT2/SLC20A2, leads to their fusion with cells expressing SynB, with no cross-reactive fusion activity with SynA. The interaction between the two partners was further demonstrated by immunoprecipitation. PiT1/PiT2 chimera and truncation experiments identified the PiT1 N-terminus as the major determinant for SynB-mediated fusion. RT-qPCR analysis of PiT1 expression on a panel of mouse adult and fetal tissues revealed a concomitant increase of PiT1 and SynB specifically in the developing placenta. Finally, electron microscopy analysis of the placenta of PiT1 null embryo before they die (E11.5) disclosed default of ST-II formation with lack of syncytialization, as previously observed in cognate SynB null placenta, and consistent with the present identification of PiT1 as the SynB partner.

IMPORTANCE

Syncytins are envelope genes of endogenous retroviruses, coopted for a physiological function in placentation. They are fusogenic proteins that mediate cell–cell fusion by interacting with receptors present on the partner cells. Here, by devising an
in vitro
fusion assay that enables the screening of an ORFeome-derived expression library, we identified the long-sought receptor for syncytin-B (SynB), a mouse syncytin responsible for syncytiotrophoblast formation at the fetomaternal interface of the mouse placenta. This protein – PiT1/SLC20A1 – is a multipass transmembrane protein, also known as the receptor for a series of infectious retroviruses. Its profile of expression is consistent with a role in both ancestral endogenization of a SynB founder retrovirus and present-day mouse placenta formation, with evidence—in PiT1 knockout mice—of unfused cells at the level of the cognate placental syncytiotrophoblast layer.
Salmonella
associated with agricultural animals exhibit diverse evolutionary rates and show evidence of recent clonal expansion
Authors: Chen Ruixi , Yang Linghuan , Pajor Magdalena S. , Wiedmann Martin , Orsi Renato H. 
Published: September 2024

ABSTRACT

Most foodborne salmonellosis outbreaks are linked to agricultural animal products with a few serovars accounting for most
Salmonella
isolated from specific animal products, suggesting an adaptation to the corresponding animal hosts and their respective environments. Here, we utilized whole-genome sequence (WGS) data to analyze the evolution and population genetics of seven serovars frequently isolated from ground beef (Montevideo, Cerro, and Dublin), chicken (Kentucky, Infantis, and Enteritidis), and turkey (Reading) in the United States. In addition, publicly available metadata were used to characterize major clades within each serovar with regard to public health significance. Except for Dublin, all serovars were polyphyletic, comprising 2–6 phylogenetic groups. Further partitioning of the phylogenies identified 25 major clades, including 12 associated with animal or environmental niches. These 12 clades differed in evolutionary parameters (e.g., substitution rates) as well as public health relevant characteristics (e.g., association with human illness, antimicrobial resistance). Overall, our results highlight several critical trends: (i) the
Salmonella
generation time appears to be more dependent on source than serovar and (ii) all serovars contain clades and sub-clades that are estimated to have emerged after the year 1940 and that are enriched for isolates associated with humans, agricultural animals, antimicrobial resistance (AMR), and/or specific geographical regions. These findings suggest that serotyping alone does not provide enough resolution to differentiate isolates that may have evolved independently, present distinct geographic distribution and host association, and possibly have distinct public health significance.

IMPORTANCE

Non-typhoidal
Salmonella
are major foodborne bacterial pathogens estimated to cause more than one million illnesses, thousands of hospitalizations, and hundreds of deaths annually in the United States. More than 70% of
Salmonella
outbreaks in the United States have been associated with agricultural animals. Certain serovars include persistent strains that have repeatedly contaminated beef, chicken, and turkey, causing outbreaks and sporadic cases over many years. These persistent strains represent a particular challenge to public health, as they are genetically clonal and widespread, making it difficult to differentiate distinct outbreak and contamination events using whole-genome sequence (WGS)-based subtyping methods (e.g., core genome allelic typing). Our results indicate that a phylogenetic approach is needed to investigate persistent strains and suggest that the association between a
Salmonella
serovar and an agricultural animal is driven by the expansion of clonal subtypes that likely became adapted to specific animals and associated environments.
LincR-PPP2R5C deficiency enhancing the fungicidal activity of neutrophils in pulmonary cryptococcosis is linked to the upregulation of IL-4
Authors: Yang Chen , Gong Ying , Liu Shan , Sun Chunan , Wang Ting , Chen Xin , Liu Wei , Zhang Xia , Yang Yonglin , Zhang Mingshun 
Published: September 2024

ABSTRACT

Pulmonary cryptococcosis is a common complication in immunocompromised patients. In a mouse model of pulmonary cryptococcosis,
Cryptococcus neoformans
induces a type 2 immune response that is detrimental to host protection. Long non-coding RNAs (lncRNAs) have emerged as key players in the pathogenesis of infectious diseases. However, the roles and mechanisms of lncRNAs in fungal infection are largely elusive. In the present study, we aimed to explore the roles of LincR-PPP2R5C in pulmonary cryptococcosis. We observed an increase in the level of LincR-PPP2R5C in the lung tissues of C57BL/6J mice after tracheal infection with
C. neoformans
. Subsequently, we intratracheally infected LincR-PPP2R5C knockout (KO) mice and wild-type mice with
C. neoformans
. LincR-PPP2R5C deficiency mitigates
C. neoformans
infection, which can be demonstrated by extending survival time and decreasing fungal burden in the lung. In the lung tissues of infected LincR-PPP2R5C KO mice, there was a notable increase in the levels of type 2 cytokines [interleukin (IL)-4 and IL-5] and an increase in the number of neutrophils in both the lung tissue and bronchoalveolar lavage fluid. Mechanistically, the lack of LincR-PPP2R5C results in increased protein phosphatase 2A phosphorylation, thereby enhancing the fungicidal activity of neutrophils against
Cryptococcus neoformans
, with IL-4 playing a synergistic role in this process. Overall, LincR-PPP2R5C deficiency mitigated pulmonary cryptococcosis by increasing the fungicidal activity of neutrophils, which was associated with increased IL-4 levels. Our study presented specific evidence of the role of host-derived lncRNAs in the regulation of
C. neoformans
infection.

IMPORTANCE

Pulmonary cryptococcosis is a human fungal disease caused by
Cryptococcus neoformans
, which is common not only in immunocompromised individuals but also in patients with normal immune function. Therefore, studying the control mechanisms of pulmonary cryptococcosis is highly important. Here, we demonstrated that the deletion of LincR-PPP2R5C leads to increased killing of
C. neoformans
by neutrophils, thereby reducing pulmonary cryptococcal infection. These findings will greatly enhance our understanding of the mechanisms by which lncRNAs regulate the pathogenesis of
C. neoformans
, facilitating the use of lncRNAs in pulmonary cryptococcosis therapy.
Development of novel multi-protein chimeric immunogens that protect against infection with the Lyme disease agent,
Borreliella burgdorferi
Authors: O’Bier Nathaniel S. , Camire Andrew C. , Patel Dhara T. , Billingsley John S. , Hodges Kelly R. , Marconi Richard T. 
Published: September 2024

ABSTRACT

Lyme disease is the most common tick-borne disease in North America. A vaccine for use in humans is not available. Here, we detail the development of two chimeric vaccine antigens, BAF and Chv2M. BAF elicits Abs that target proteins and protein variants produced by
Borreliella
species in ticks (OspB and OspA) and mammals (FtlA/B). OspB serves as the backbone structure for the BAF chimeric. Two OspA
221-240
epitope-containing domain (ECD) variants (#A1 and #A15) were inserted into a loop in OspB. The N-terminal region of the FtlA protein was joined to the C-terminus of the chimeric. The second chimeric, Chv2M, consists of L5 (loop 5) and H5 (helix 5) ECDs derived from diverse OspC proteins.
Borreliella
species produce OspC upon exposure to the bloodmeal and during early infection in mammals. Here, we demonstrate that BAF and Chv2M are potent immunogens that elicit Abs that bind to each component protein (FtlA, FtlB, OspB, and multiple OspA and OspC variants). Anti-BAF and anti-Chv2M Abs kill
Borreliella burgdorferi
strains through Ab-mediated complement-dependent and complement-independent mechanisms. Eighty percent (32/40) of mice that received a three-dose vaccine regimen were protected from infection with
B. burgdorferi
B31. Efficacy increased to 90% (18/20) when the amount of Chv2M was increased in the third vaccine dose. Readouts for infection were
flaB
PCR and seroconversion to VlsE. This study establishes proof of principle for a chimeric immunogen vaccine formulation that elicits Abs to multiple targets on the
B. burgdorferi
cell surface produced during tick and mammalian stages of the enzootic cycle.

IMPORTANCE

Lyme disease is a growing public health threat across parts of the Northern Hemisphere. Regions that can support sustained tick populations are expanding, and the incidence of tick-borne diseases is increasing. In light of the increasing risk of Lyme disease, effective preventive strategies are needed. Most vaccine development efforts have focused on outer surface protein A, a
Borreliella burgdorferi
protein produced only in ticks. Herein, we describe the development of a novel vaccine formulation consisting of two multivalent chimeric proteins that are immunogenic and elicit antibodies with bactericidal activity that target several cell surface proteins produced by the Lyme disease spirochetes in feeding ticks and mammals. In a broader sense, this study advances efforts to develop custom-designed vaccinogens comprised of epitope-containing domains from multiple proteins.