Endocytosis is a fundamental mechanism by which cells perform housekeeping functions. It occurs via a variety of mechanisms and involves many regulatory proteins. The GTPase dynamin acts as a “molecular scissor” to form endocytic vesicles and is a critical regulator among the proteins involved in endocytosis. Some GTPases , membrane proteins , and secondary messengers  mediate dynamin-independent endocytosis. These pathways may be convergent, as multiple pathways exist in a single cell. However, what determines the specific path of endocytosis is complex and challenging to comprehend. This review summarizes the mechanisms of dynamin-independent endocytosis, the involvement of microRNAs, and factors that contribute to the cellular decision about the specific route of endocytosis. Endocytosis is an evolutionarily conserved cellular process in all eukaryotic cell types . BiologiThough dynamin’s role in cellular endocytosis has been well studied at the molecular level, recent studies shed new light on dynamin-independent internalization and trafficking of endosomes ,19. The In this review, we discuss dynamin-independent endocytosis mechanisms, the role of microRNAs (miRNAs), and factors that determine the endocytosis (dynamin-dependent/independent) mechanisms.Endocytosis of GPI-APs reflects a unique internalization route. It is clathrin- and dynamin-independent, and the cargo is routed into GPI-AP-enriched early endosomal compartments (GEECs) by integration of uncoated tubulovesicular clathrin-independent carriers (CLICs) emanating directly from the plasma membrane . Small GADP-ribosylation factors (Arfs) are Ras family GTPases that are classified into three families . Class IFlotillins are evolutionarily conserved membrane-associated proteins and include flotillin-1 and flotillin-2. They are ubiquitously expressed in mammalian tissues and regulate many cellular functions ranging from cell signaling, regulation of the cortical cytoskeleton, endocytosis, and protein trafficking to gene expression ,50. The 2). These results suggest that CaSR-mediated ERK1/2 signaling occurs in a dynamin-independent manner [2+ influx, respectively. In neuronal cells, the neuronal activity and cell signaling pathways such as PKA activation are regulated by intracellular calcium [Calcium plays a vital role in synaptic transmission and internalization of membrane vesicles following neurotransmitter release via exocytosis . The keyt manner . Studies calcium . A disti calcium ,72,73. L calcium . Taken tPinocytosis is a process of cell drinking where the cell internalizes extracellular fluid via endocytosis . PinocytMicropinocytosis takes part in several cellular functions. The uptake of thyroglobulin (Tg) occurs through micropinocytosis, where Tg rapidly fuses with primary lysosomes and releases iodinated compounds upon subsequent maturation . It is aMacropinocytosis is an actin-driven endocytic process and it can be either a signal-dependent process (occurs in response to growth factor stimulation) ,84 or a Entosis is a non-apoptotic cell death program with a cell-in-cell (CIC) cytological feature. It is often referred to as a cannibalistic quality of cells, where those detached from adherent junctions are engulfed by other healthy cells . In contUnder physiological conditions, entosis prevents the accumulation of unhealthy cells. For instance, entosis prevents aneuploidy in a p53-dependent manner during mitosis. Generation of an abnormal cell or DNA damage during prolonged metaphase in mitosis triggers the activation of tumor suppressor p53, which drives cell-in-cell formation via Rnd3-compartmentalized RhoA activity . FurtherThere are distinct yet-to-be-characterized endocytic mechanisms that are regulated by a dynamin-independent mechanism. For example, a pinocytic chaperone pincher is involved in the internalization of myelin-associated inhibitors for axonal growth Nogo-A in neurites. Nogo-A internalization is associated with neuronal growth arrest via the cAMP pathway . AnotherMicroRNAs (miRNAs), also known as small non-coding RNAs, are widely recognized for regulating gene expression, which modulates physiological and pathological processes. However, their role in endocytosis and intracellular trafficking remains largely unknown. Over the past decade, research on miRNAs has shown their involvement in endocytosis processes ,104,105.Dynamin-independent endocytosis mechanisms are widely implied to regulate physiological functions and pathological processes. Several nutrients and cellular components are internalized through endocytosis and affect cellular physiology. For example, Arf6-mediated endocytosis is crucial for nutrient and ion transport, cell–cell interactions , and immunological functions  . Folate Endocytosis is a complex biological trafficking process within cells that attracts considerable therapeutic interest, as dysregulation of endocytosis has been linked to the pathogenesis of several diseases. In astrocytes, the dynamin-independent endocytosis by Rab5 significantly upregulated an amyloid-β-peptide and thus can be further explored as a therapeutic target in Alzheimer’s disease . SimilarThe dynamic plasma membrane hosts numerous endocytic pathways. While existing data suggest distinct molecular machinery and their cargo specificity in internalization and trafficking during endocytosis, several questions remain unanswered regarding its diversity, complexity in molecular machinery, and choice of internalization route in a divergent cellular context. The endocytosis fate of cargo cannot be generalized, and it can be varied depending on the type of internalization, extracellular environment of the cell, pathophysiological status, cell type, receptor subtype, etc. In the context of receptor trafficking, there have been several instances where a single receptor can undergo internalization via both dynamin-dependent and independent pathways. For example, internalization of EGFR through a clathrin-dynamin-dependent mechanism recycles the receptor to the plasma membrane and prolongs EGFR activity, which may have a role in cell proliferation and cancer cell growth . In contCollectively, dynamin-independent endocytic mechanisms play important roles in multiple processes ranging from physiological functions to pathological conditions. Of note, unraveling the pathways that intersect dynamin-dependent and -independent endocytosis is challenging and often leads to a common molecular outcome. The use of advanced molecular tools involving single-cell genomic studies and characterization of the endocytic pathway in the context of specific cargo being routed and their trafficking could provide helpful insights in dissecting the dynamin-independent endocytic biology at the molecular level. Furthermore, miR’s role in endocytic regulation is emerging, and determining their role in signaling and the functional outcome has therapeutic importance. Taken together, understanding these mechanisms of endocytosis might offer disease-modifying strategies and serve as a valuable tool for designing novel therapeutic candidates for such diseases.


Mental disorders among adolescents represent a high burden and early onset. They compromise their physical health, survival, and future potential. On the other hand, young people have inadequate access to essential health services in sub-Saharan Africa. We aimed to review school-based psychological interventions, contents, delivery, and evidence of effectiveness designed to treat depression, anxiety, or posttraumatic stress symptoms among adolescents and young adults aged 10–24.We searched articles on the following databases: PubMed, Scopus, Embase, and Science Direct from 17/10/2022 to 30/12/2022. Furthermore, relevant studies were searched from advanced google scholar, google and identified reference lists. We used MeSH browser for key words: psychological interventions, depression, anxiety, posttraumatic stress disorder and lists of Sub-Saharan Africa countries. We combined words using standard Boolean operators . The quality of studies was evaluated using the Cochrane Collaboration’s risk of bias tool and the results were presented as a narrative synthesis since the interventions were very heterogenous.Fourteen randomized controlled trials were included for systematic review and more than half (57.14%) were from Kenya and Nigeria. Common school-based psychological interventions were cognitive behavioral therapy and Shamiri interventions . More than half (57.14%) of the interventions were delivered by non-specialists like teachers, lay providers and community health workers. Nearly one-fifth of the interventions were used individual modality. School-based psychological interventions provided by non-specialists also produced a greater reduction in adolescents’ depressive, anxiety, and post-traumatic stress symptoms compared to the control groups.Cognitive behavioral therapy and Shamiri interventions were the common treatment delivered in school settings. The range of interventions could be effectively delivered by non- professionals that promote task-shifting of psychological interventions from very scarce mental health specialists in these countries.Trial Registration: ProsperoCRD42022378372. https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022378372. Mental disorders are responsible for a large proportion of burden of disease among adolescents in all societies. Most mental health conditions begin early in life (12–24 years of age): 50% arising before age 14 and 75% by 24, even though they are often first identified later in life [According to the Lancet Child and Adolescent Health published in 2018, the definition of adolescent age is more expanded and inclusive from 10–19 years to 10–24 years. This is the delayed role transitions that include completion of education, marriage, and parenthood shifts familiar perceptions of when adulthood begins. The age of 10–24 years more closely to adolescent growth and understandings of the life phases and facilitate extended investments across a wide range of settings. It is essential for developmentally appropriate framing of laws, social policies, and service systems .The Sub-Saharan Africa (SSA) has the highest rate of disability-adjusted life years and the fastest-growing adolescent population in the world . HoweverMental disorders among adolescents represent the main threat to their health, survival, and future potential worldwide . The mosDespite the high burden and impacts, most mental health conditions remain unrecognized and untreated . Many AfIntervention development and adaptation have been primarily focused on HIV/AIDS service delivery in school or community programs by non-specialist health workers. There is a critical need for child and adolescent mental specialist, integration of those services into primary healthcare, coordinated cross-sectoral cooperation, and established referral networks . EvidencPsychological interventions in school enhance academic achievement by addressing self-esteem and social well-being . AdolescThere are trials of school-based psychological interventions carried out in SSA countries among adolescents with the need for summarized relevant studies to provide a better treatment appropriate to the culture. The studies were too heterogenous to pooled using meta-analysis in terms of psychological intervention in principles, duration of therapy, session duration, delivery mode , content, different approaches, and outcome measurements. One of the recommendations by Cochrane Handbook, the results will not be pooled using meta-analysis if there are substantial heterogeneity found because it may produce misleading results . TherefoThis systematic review focused only on randomized controlled trials (RCTs) since they have a lower risk of bias compared to other study designs. Therefore, the findings from this review provide the basis for practitioners working to improve the mental health of adolescents, policy planners, and researchers involved in developing and delivering school-based mental health initiatives.The review aimed to assess school-based psychological interventions’ contents, delivery and evidence of effectiveness designed to treat depression, anxiety, or PTSD among adolescents aged 10–24.We used the PICO framework for the systematic review question: are school-based psychological interventions (intervention) effective for adolescents (population) compared to usual or other care (comparison) on depressive, anxiety, or post-traumatic stress symptoms (outcome) in SSA)?We used the Cochrane Handbook for the systematic review of interventions version 6.3 and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) 2020 guidelines  See S1 . We usedArticles eligible for inclusion were required to demonstrate the following characteristics:Population: participants were adolescents (10–24 years). This is a broader and more encompassing definition of adolescence to frame laws, social policies, and service systems in a manner that is developmentally appropriate [ropriate .Intervention: school-based psychological interventions conducted in SSA and designed to reduce depressive, anxiety and PTSD symptomsComparison: control group that include no intervention, usual care, alternative intervention, or waitlistOutcome: The psychological treatment outcomes included depression, anxiety, or post-traumatic stress disorder (PTSD)Study design: Randomized controlled trials conducted in SSA. The restriction of SSA intended to narrow down the scope of review to countries that share similar socio-economic, cultural, and political situations.Context: Published in English language (due to limitation of others language by the team) from 2010–2022. The year 2010 was also selected because WHO launched the Mental Health Gap Action Program (mhGAP) to scale up care and services using evidence-based interventions for preventing and managing mental illness, including child and adolescents mental health problems [problems .There were exclusion criteria:Health institutions-based interventionsStudies that did not measure depression, anxiety or PTSD as an outcomeStudies that included adults and adolescents without presenting results separately by ageStudies evaluated mixed interventions, such as both psychological and pharmacological interventions.Trials of interventions delivered in universities or colleges. We believed that all college/university students in SSA not under the age of 24 years and the nature of stressors somewhat different from secondary school students.In the beginning, index terms and keywords were identified based on the participants, intervention, comparator and outcome (PICO) framework . We systWe used MeSH browser for key words: psychological interventions, depression, anxiety, posttraumatic stress disorder and lists of Sub-Saharan Africa countries. We combined words using standard Boolean operators . The key terms used to retrieve primary articles were adolescent* OR youth* OR teen* OR “young people” AND depression OR “depressive symptoms” OR “depressive disorder” OR “affective disorder” OR “major depression” OR “major depressive disorder” OR “probable depression” OR “anxiety disorders” OR “social anxiety” OR anxiousness OR anxious OR anxiety OR “internalized problems” OR “emotional problems” OR “post-traumatic” OR posttraumatic OR PTSD OR “psychological distress” AND “group psychological interventions” OR “brief psychosocial interventions” OR “psychosocial interventions” OR “psychological interventions” OR “psychological treatment” OR “psychological therapy” OR “psycho-supportive interventions” OR psychoeducation OR “self-help intervention” OR “stress management” OR “emotional support” OR “interpersonal therapy” OR “behavioral intervention” OR “behavioural activations” OR “cognitive behavioural therapy” OR “cognitive behavioral therapy” OR “problem solving” OR “group therapy” OR “social skill interventions” OR psychotherapy OR counseling OR mindfulness AND lists of Sub-Saharan Africa countries see .We removed duplicate articles using endnote (version 20.0.0.14672) before starting the two-stage selection process. Two independent authors (MT and BA) read study titles and abstracts based on eligibility criteria and classified references: yes (included), maybe (included) and no(excluded). Any disagreement was resolved via consensus or by involving a third author (TW). Abstracts in which eligibility was unclear underwent full-text review. Then, the remaining full-text articles were assessed for inclusion by two authors (MT and BA). Furthermore, relevant studies were searched from identified reference lists manually.Two reviewers critically appraised the internal validity of eligible studies using the Cochrane Collaboration’s risk of bias tool (RoB 2) . The RoBWe used data extracted based on recommendations by the Cochrane Handbook for Systematic Reviews of Interventions. Two reviewers (MT and BA) extracted data independently using a data extraction form that includes: the name of the first author, year of publication, country of study, age of participants, sample size, type of intervention, number of sessions, length, comparison group and study outcomes, the effect of the interventions. Data were extracted into a Microsoft Excel spreadsheet See .Search results were summarized in the PRISMA 2020 flow diagram. Out of 3033 articles retrieved, 66 articles were removed due to duplication through EndNote 20 citation manager. Then, 2967 papers were excluded after the title and abstract screening based on inclusion criteria. The remaining forty-one full texts were checked in detail for inclusion criteria again and only fourteen RCTs were included for systematic review. The main reasons for twenty-seven full text exclusions were: not school-based  studies, out of SSA, out of age range, not randomized controlled trials, including pharmacological intervention and not reported depression, anxiety, or PTSD as an outcome .From the included RCTs, only three were found to have a low risk of bias –33. The Overall, 4262 individuals were represented from seven countries in SSA. From fourteen included studies, the highest number reported from Kenya (four) ,33,37–39The details of intervention characteristics include content, mode of delivery, duration, number of sessions, who delivered the intervention, and the control group found in the More than half (n = 8) of the interventions were delivered by non-specialists such as teachers ,40, lay The number of intervention sessions among fourteen RCTs ranging from single to 16 sessions. One session length ranged from the shortest 30 minutes to a maximum of 90 minutes. Follow-up durations of interventions across the included studies ranged from one week ,40,43 toBased on pre- and post-intervention assessments, the effectiveness of psychological interventions in reducing the symptoms of depression, anxiety, or PTSD was determined. Post-intervention results showed a significant reduction in symptoms for depression ,38,40–42Fourteen RCTs were conducted on the effectiveness of school-based psychological interventions after contextual adaptations from seven countries in this region, the majority (71.4%) were from Kenya, Nigeria, and South Africa. The most common psychological interventions were CBT  and Shamiri intervention . In general, psychological interventions provided in school settings were effective in reducing depressive, anxiety and posttraumatic stress symptoms when compared to the controlled groups based on pre-and post- interventions measures.There was considerable variation in the school-based psychological interventions, modality, intensity of the session, facilitators expertise, duration of interventions and follow-up period to address the commonest mental health problem of adolescents. In our review, the psychological interventions were delivered by different professionals such as psychiatrists, social workers, guidance counselors, Nurses, and teachers. Interventions delivered by trained non-specialists also found a reduction in depressive, anxiety or traumatic stress symptoms. This might be non-specialists can be easily accessible, cost-effective and reduce stigma for majority of adolescents. A meta-analysis revealed that teacher-delivered interventions were more effective than controls in reducing depressive, anxiety, and posttraumatic stress symptoms in students ,46. In sSessions numbers used range from single session to 16 sessions, while the follow-up period is from one week to two years. This is consistent with a systematic review among adolescents suggesting that school-based psychological interventions were successful in reducing mental health problems  and sympIn this review, the most frequently utilized intervention was CBT. Studies that utilized CBT in this review, found significant reductions in depressive or traumatic stress symptoms. This is consistent with previous study findings that reported that CBT delivered to young people in schools can reduce symptoms of depression and anxiety ,50,51. AIn this review, Shamiri (a Swahili word for thrive) was the second most common school-based intervention. The Shamiri intervention teaches youths to cultivate a growth mindset, practice gratitude, and take value-aligned action. Four RCTs in Kenya have shown the intervention effectively lowers youth mental health issues like depression and anxiety while enhancing social interactions, academic performance, school atmosphere, and other functioning outcomes ,33,37,38The majority of interventions in these reviewed articles used a group-based modality. This might be because group therapy became effective for interpersonal and intrapersonal psychological problems. Group therapy can provide many possible advantages that individual therapy does not: understanding of the universality of the problem, improved communication skills, forming relationships, social skills, improved self-esteem, and increased coping skills . A meta-The improvement was significantly better than control group and depressive, anxiety, or PTSD symptoms were significantly lower within a month of completing psychological therapy . The difThis review can be used as baseline evidence for the Ministry of Health and Education, school leaders, teachers, health professionals, and psychologists when selecting evidence-based interventions to support students’ mental health and wellbeing in Sub-Saharan countries and in LMICs with similar socio-economic status. Psychological interventions in school settings are effective at reducing depression, anxiety, and trauma symptoms. This emphasizes the significance of providing mental health services in educational settings since many students experience a range of mental health problems that may affect their achievement and wellness in terms of their academic, relational, social, and physical well-being. Of note, teacher-delivered interventions in school could be less stigmatized, easily accessible, cost-effective, and affordable for most adolescents to fill the large mental health care gap. This in turn, alleviate the four main barriers of adolescents not seeking and accessing help for their mental health problems identified in the previous systematic review. These include, individual factors , social factors , perceptions of the therapeutic relationship , and systemic and structural barriers  . In addiOur reviews showed that school-based psychological interventions significantly reduce depressive, anxiety, or PTSD symptoms among adolescents. However, there were several limitations in this review. First, we advise the generalizability of this finding to SSA in caution since there was the over-representation of two countries, under-representation of others, and no representation for most of the countries in SSA. Second, the intervention delivery format, duration of interventions, sessions intensity, and follow up length varied among the included studies. Therefore, to bring a single conclusion on the above-mentioned variability of interventions could not be drawn. Future reviews better examine how these variabilities of interventions may influence their effectiveness. In addition, a few trials found a low risk of bias and nearly half of them were not registered during the quality assessment.This systematic review provides evidence on school-based psychological interventions for managing common adolescent mental health problems such as depression, anxiety, and posttraumatic stress disorders in SSA. The review showed school-based psychological interventions had a stronger impact on symptom reduction. The most commonly used interventions from the included studies were CBT and non-CBT approaches. The range of interventions could be effectively delivered by non-professionals such as teachers, per-lead or community leaders that promote task-shifting of psychological interventions from very scarce mental health specialists in these countries. Particularly, psychological interventions provided in schools by teachers or peer-led facilitators can be more affordable, less stigmatized, and easily accessible for the vast majority of adolescents. This makes the interventions could be more scalable as well as sustainable to address the mental health need of adolescents. This, in turn, narrows the very large mental health treatment gaps in SSA, helps the students improve functioning at school, and reduces the distress of the student, teachers, and parents.S1 Checklist(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Details of the catalytic mechanism haveremained under debate and, to date, synthetic model complexes of theCuZ*/CuZ sites are extremely rare due to thedifficulty in building the unique {Cu4(μ4-S)} core structure. Herein, we report the synthesis and characterizationof [Cu4(μ4-S)]n+  clusters,supported by a macrocyclic {py2NHC4} ligand, inboth their 0-hole (2) and 1-hole (3) states,thus mimicking the two active states of the CuZ* site duringenzymatic N2O reduction. Structural and electronic propertiesof these {Cu4(μ4-S)} clusters are elucidatedby employing multiple methods, including X-ray diffraction (XRD),nuclear magnetic resonance (NMR), UV/vis, electron paramagnetic resonance(EPR), Cu/S K-edge X-ray emission spectroscopy (XES), and Cu K-edgeX-ray absorption spectroscopy (XAS) in combination with time-dependentdensity functional theory (TD-DFT) calculations. A significant geometrychange of the {Cu4(μ4-S)} core occursupon oxidation from 2 (τ4(S) = 0.46,seesaw) to 3 (τ4(S) = 0.03, square planar),which has not been observed so far for the biological CuZ(*) site and is unprecedented for known model complexes. The singleelectron of the 1-hole species 3 is predominantly delocalizedover two opposite Cu ions via the central S atom, mediated by a π/πsuperexchange pathway. Cu K-edge XAS and Cu/S K-edge XES corroboratea mixed Cu/S-based oxidation event in which the lowest unoccupiedmolecular orbital (LUMO) has a significant S-character. Furthermore,preliminary reactivity studies evidence a nucleophilic character ofthe central μ4-S in the fully reduced 0-hole state.The active site ofnitrous oxide reductase (N The rtransfer1a.16−23 activity1a.214(μ4-S)} coresof both restingCuZ* and CuZ sites adopt a seesaw geometry witha large CuI–S–CuII angle of ∼160° dx2−y2 and the S 3py orbitals,thus leading to a CuI–S–CuII σ/σ superexchangepathway for electron delocalization in the 1-hole CuZ*form.18 Electron paramagnetic resonance (EPR) andmagnetic circular dichroism (MCD) spectroscopies evidenced that thespin density of 1-hole CuZ* is delocalized unevenly overthe two Cu ions (∼5:2),18 whereas the spin populationis evenly localized on two and three Cu ions for the 1-hole CuZ° and CuZ, respectively,24 indicating significant structural differences between these states.Spectroscopic methods in combination with theoretical calculationshave shown that the additional coordination site at the CuI–CuIVedge may be vacant (for 0-hole CuZ*) or may be occupiedby a μ2–OH (for resting CuZ*),25 terminal CuIV–OH (for CuZ°),23 μ2-S2– (for CuZ),15 and μ2-SH– (for 1-hole CuZ).24 Despitethe apparent distinctions in the edging ligands, the {Cu4(μ4-S)} core structures were so far assumed to belargely unchanged, mostly on the basis of computational models thatused truncated structures derived from the parental resting species,which also satisfies the requirement for a minimal reorganizationenergy during ET processes.24 Nevertheless, due tothe lack of crystallographic data of N2ORs where the CuZ*/CuZ site is in a nonresting state, the structuralproperties of CuZ* (1-hole), CuZ°, andCuZ (1-hole), especially their {Cu4(μ4-S)} core structures, are yet to be investigated. Gainingexperimental information about geometric and electronic structurevariations during redox interconversion of the {Cu4(μ4-S)} unit would thus be valuable and might contribute to theunderstanding of the catalytic mechanism of N2O reduction.The {Cuof ∼160° 1a,13,15 4(μ4-S)} core structureand the rich redox chemistry of the CuZ*/CuZ site have stimulated many efforts to synthesize model complexeswith such a motif, not only as an alternative way to study the short-livedbiological intermediates but also to exploit the use of such metalclusters in, e.g., photochemistry and catalysis.28 However, buildinga tetranuclear copper core bearing a single S-cap has proven extremelychallenging due to the propensity to form copper clusters with multipleS atoms.32 So far, only very few model complexes containing the {Cu4(μ4-S)} motif have been reported, and their supportingligands are limited to bidentate phosphine ligands  or amidinate ligands (NCHN–). The first synthetic example of a {Cu4(μ4-S)} cluster was reported by Yam et al. in 1993 before the structureof N2OR was elucidated; hence, it was not discussed inthe context of the biological site.34 Its {Cu4(μ4-S)} core, which was isolated solely inthe [4CuI] state, was supported by a diphosphine ligand(dppm) and featured a square pyramidal geometry (A). After Yam’swork, some di- and tricopper clusters bridged by a single S atom werereported,37 but the second example of a synthetic {Cu4(μ4-S)} cluster appeared only in 2014.38 Modifying the dppm ligand, Mankad et al. isolateda {Cu4(μ4-S)} cluster with NH-bridgedbidentate phosphorus ligands , yet again only in the[4CuI] state. Interestingly, this complex showed stoichiometricN2O reduction reactivity under very specific reaction conditions,which was proposed to be supported by the secondary coordination sphereN–H groups that could serve as hydrogen bond donors.39 A seminal breakthrough was made by the samegroup when the neutral diphosphine ligands were replaced by anionicamidinate ligands (NCHN–), and a redox pair of {Cu4(μ4-S)} clusters in the [2CuI:2CuII] 40 and [3CuI:CuII] 41 states was obtained. The {Cu4(μ4-S)}cores in both states adopt square pyramidal geometries with ratherbent Cu–S–Cu (opposite Cu ions) angles of up to 126°.EPR spectroscopy showed that the unpaired electron of the 1-hole speciesis delocalized evenly over the four Cu ions. At low temperature (−78°C), N2O can be stoichiometrically reduced by the1-hole species to N2 and O2– along withthe formation of the 2-hole species.42The unique {Cugeometry 1b, A. Afds dppa; 1b, B, ye 2-hole; 1b, C40 a 1-hole, 1b, D41 sZ siteis still very limited, with an obvious scarcity of ligand scaffoldsthat can stabilize {Cu4(μ4-S)} clustersin different states. In particular, a system that allows for the isolationand investigation of a pair of {Cu4(μ4-S)} complexes in the biologically relevant [3CuI:CuII]  and [4CuI]  states is still lacking. N-Heterocycle carbenes (NHCs) are increasingly employedas alternative ligands and as surrogates of histidine residues inbioinspired model chemistry, facilitating the stabilization of reactiveintermediates and unusual oxidation states.49 In our recent work, we found that the macrocycles {py2NHC2}  and {py2NHC4} (L), featuring combinations of pyridine and NHC donors,show great flexibility and can support mononuclear complexes [L’Cu]1/2/3+ in various Cu oxidation states 51 as well as binuclear complexes [LCu2]2/3+ inthe CuICuI and mixed-valent Cu1.5Cu1.5 states,52 respectively.The mixed-valent complex [LCu1.5Cu1.5]3+ features a large spin delocalization energy and fast electron self-exchangerate, which structurally and functionally mimics the CuA site. In the present study, novel {Cu4(μ4-S)} clusters scaffolded by the macrocycle L were isolated and fullycharacterized in both the 0-hole and 1-hole states, i.e., in the activestates of the CuZ* site during the N2O reductionprocess (4(μ4-S)} clusters reported so far, and a significant redox-inducedstructural change in the {Cu4(μ4-S)} corehas now been evidenced by X-ray diffraction (XRD) analyses. The correspondingelectronic structure changes have been analyzed by a range of spectroscopictechniques, including UV–vis, EPR, Cu K-edge X-ray absorptionspectroscopy (XAS), and Cu/S X-ray emission spectroscopy (XES), andthe experimental results were correlated with DFT calculations.In spiteof the significant achievements made by the Yam and Mankadgroups, the modeling chemistry for the unique Cuprocess 1a,c.The2-S-bridged neutral dicopper species [LCu2(μ2-S)] that could mimic a fragment of thecore structure ofthe CuZ*/CuZ site. However, treating the dicoppercomplex [LCu2](PF6)2 (1) with 1 equiv of sodium sulfide (Na2S) in acetonitrile(MeCN) gave, after workup and slow diffusion of diethyl ether (Et2O) into the MeCN solution, the μ4-S-cappedtetranuclear copper complex, [L2Cu4(μ4-S)](PF6)2 (2) as yellowcrystals (2 has a {Cu4(μ4-S)} core supported by two macrocyclic ligands L (1. Two Cu ions(Cu2 and Cu4) in opposite positions are coordinated by NHCs from thesame ligand (L) but different {pyNHC} cavities, while the other twoopposite Cu ions (Cu1 and Cu3) are coordinated by NHCs from differentligands L, thus rendering two pairs of Cu ions in distinct coordinationenvironment (Cu2 and Cu4 vs Cu1 and Cu3). In contrast, in all otherCuZ* model complexes reported so far, neighboring Cu ionsof the {Cu4(μ4-S)} core are spanned bybidentate phosphine or amidinate ligands in the same coordinationmode (A–D).41 The Cu–S bonddistances in 2 are found in the narrow range 2.3021(6)–2.3499(5)Å and are similar to the Cu–S distances in the CuZ*/CuZ sites of N2OR.15 The projection of the Cu4 base is an irregular quadrilateral,with Cu···Cu distances varying from 2.972(1) to 3.370(1)Å (4-S1)–Cu3 angle of 160.80(3)o is comparable to that in the CuZ*/CuZ sites (∼160°), while the Cu2–(μ4-S1)–Cu4 angle of 134.71(3)o is larger than thatin the biological systems (∼90 o). The τ4 value is 0.46 for the μ4-sulfide ligandin 2, indicating a seesaw shape of the {Cu4(μ4-S)} core ,53 which issmaller than τ4(μ4-S) in the CuZ*/CuZ sites (0.74/0.71).15 Notably, this τ4(μ4-S) value isthe smallest among the reported CuZ*/CuZ modelcomplexes (0.59–0.90), in which the {Cu4(μ4-S)} core is best described as a square pyramid with morebent Cu−μ4-S–Cu angles . Interestingly, the molecular structureof the cation of 2 in the solid state has an approximate C2 local symmetry. However, 1H NMRspectra in different solvents  show only one set of signals in the temperature range studied with equivalent protons of both halves of the macrocyclicligand (L), implying a higher level of symmetry in solution. Thismight be caused by the fast conformational inversion of the {Cu4(μ4-S)} core as well as the ligand macrocycle,leading to an averaged overall C2v symmetryon the NMR time scale.Initially, we were aiming at the synthesisof a μcrystals 1.X-ray igands L 2a. All Cionmode 1b, A–D.3370(1)Å 2b.The C2 isstable both in solidstate and in common solvents under an inert atmosphere. In MeCN solution under aerobic conditions,however, it decomposes slowly to give precursor complex 1, which likely proceeds through the oxidation of 2 byO2 and subsequent decomposition of the oxidized species(vide infra). The reformation of 1 showsthat the Cu–CNHC bonds are labile and that the rearrangementof L, interconverting the binding modes seen in 2 and 1, is reversible. The identity of 2 was furtherconfirmed by combustion analysis and by electrospray ionization massspectrometry (ESI-MS); the latter shows a dominant peak at m/z = 645.1 amu (in MeCN) with an isotopepattern that is characteristic of the dication [L2Cu4(μ4-S)]2+ , indicating that the {Cu4(μ4-S)} core structure of 2 is retained in solution.At room temperature (rt), 4(μ4-S)} cluster canbe accessed, which would emulateboth activestates of the CuZ* site that are passed through in thecatalytic N2O reduction cycle, the redox properties of 2 were examined electrochemically. Cyclic voltammetry (CV)of 2 in MeCN (100 mV/s) at rt reveals one reversibleredox event at a potential of −0.65 V , assignedto the [L2Cu4(μ4-S)]3+/2+ redox couple when a small potential range from −0.98 to −0.28V (vs Fc+/0) was scanned. This redox potential is significantlycathodically shifted compared to the 0-hole model complexes [(μ2-dppm)4Cu4(μ4-S)](PF6)2 and [(μ2-dppa)4Cu4(μ4-S)](PF6)2  for which onlyirreversible oxidation events were observed.54 This differencecan likely be attributed to the strong σ-donating ability ofthe NHC ligands; at the same time, the NHC-based scaffold L is capableof stabilizing the {Cu4(μ4-S)} core inboth relevant oxidation states. However, the redox event at E1/2 = −0.65 V becomes irreversible whenthe scan range is extended anodically to +0.42 V (vs Fc+/0), and a quasi-reversible redox event at E1/2 = −0.06 V appears (1ox/1 (E1/2 = −0.09 V).52 Thiscan be attributed to the rapid decomposition of the species resultingafter 2-fold oxidation of 2, giving two equiv of 1. In line with this interpretation, the current of the redoxevent at E1/2 = −0.06 V is approximatelytwice the current of the initial oxidation of 2 at −0.65V.To assess if the 1-holestate of this {Cu appears 3a that i2 55 at −80 °C, both absorptionsof 2 vanish and an intense broad band at λmax = 790 nm as well as a band with lower intensity at λmax = 530 nm emerge, reaching a plateau in intensity afterthe addition of 1 equiv of oxidant (2Cu4(μ4-S)](PF6)3 (3). The observation of an isosbesticpoint at 470 nm indicates a clean conversion of 2 to 3 without intermediate step(s) 55 and subsequent reoxidation with 1equiv of [Cp*2Fe]PF6 . In situ-formed 3 is stable at −35°C for several hours without noticeable decay according to UV–visspectroscopy, suggesting that the isolation of 3 shouldbe feasible at such low temperature.While the oxidized species (1-holestate) is metastableat rt,UV–vis titration experiments showed that it is relatively stableat low temperatures. The UV–vis spectrum of complex 2 3b in ace oxidant 3b. The c step(s) 3b. The i2 with [Cp*2Fe]PF6 was carried out at−35 °C in acetone } core to be establishedby X-ray diffraction analysis . The cation of 3 is structurally similar to the cationof 2 consisting of a {Cu4(μ4-S)} core, yet in an unprecedented square planar geometry where theμ4-S resides in the center of the square with a τ4(S) value of 0.03. This is in remarkable contrast to 2 and all of the other model complexes (A–D)as well as CuZ*/CuZ sites where the {Cu4(μ4-S)} cores have either seesaw shapes orsquare pyramidal shapes. To improve the crystallization behavior,counteranion exchange of 3 with NaBF4 wassubsequently performed, which resulted in high-quality purple crystalsof 3-BF4.Bulk oxidation of  acetone 1. After analysis S2. The c3-BF4 (4(μ4-S)} core as thatin 3 with a slightly larger τ4(S) valueof 0.11, which might originate from the collective packing effectsof anions (PF6– vs BF4–) and solvents (acetone vs MeCN). Upon oxidation tothe 1-hole state, the Cu4 base in 3-BF4 is less asymmetric than in 2, withCu···Cu distances in a narrower range (3.095(1)–3.265(1)Å; 2, become pairwise unequalin 3-BF4 with two long Cu–Sbonds  Å and Cu3–S1, 2.3191(9)Å) and two short Cu–S bonds Å and Cu4–S1, 2.2005(9) Å). Careful inspection ofthe structure reveals that each Cu ion adopts a trigonal planar geometry;however, the {C2NHCCuS} planes centered on Cu2and Cu4 are approximately perpendicular to the Cu4 baseplane with a dihedral angle of 86.3 and 89.0°, respectively,while the {C2NHCCuS} planes centered on Cu1and Cu3 are bisected by the Cu4 base plane with dihedralangles of 45.3 and 40.8°, respectively. Such a spatial configurationmight affect the interaction of the Cu 3d orbitals with the μ4-S 3p orbitals, resulting in the inequality of Cu–Sbond lengths in 3-BF4. Notably,the two short Cu–S bonds  are significantly contracted comparedto the corresponding Cu–S bonds in 2, suggestingthat the two opposite metal ions Cu2 and Cu4 are predominantly involvedin the oxidation process (vide infra).The structureof the cation of 3-BF4 2a featur1H NMR spectrum of 3 at 263 K displaysbroad singlets in the range of 0–18 ppm due to its paramagneticnature . The S = 1/2 ground state of 1-hole 3 was confirmed by SQUIDmagnetometry where a constant χMT value of 0.40 cm–3 mol–1 K (corresponding to μeff = 1.79 μB) over the entire temperature range from2 to 300 K was observed and best-fitted as an S =1/2 system with giso = 2.07 . The continuous-wave X-band (9.63GHz) EPR spectrum of 3 in acetone recorded at 50 K ispseudoaxial with a hyperfine pattern on the low field side attributedto the 63/65Cu (I = 3/2) nuclei, withrelatively small splittings and an intensity pattern that suggeststhe unpaired electron is delocalized over more than one Cu ion  and intensitypattern resulting from two equivalent copper centers.52 To further resolve the EPR parameters, a two-pulse echo-detectedQ-band (34.0 GHz) EPR spectrum of 3 in acetone was collectedat 15 K. This higher-frequency spectrum resolved the rhombic splittingof the g-tensor, particularly the narrow g3 feature . The overall linewidth of the Q-band spectrum is larger than the X-band spectrum, acommon occurrence for Cu centers measured at higher microwave frequencies,resulting in only a broad g1 feature withoutany well-resolved copper hyperfine coupling.56 Both the X- and Q-band spectra are best simulated with a rhombic g-tensor g =  and two equivalent Cu hyperfine tensors A(Cu) =  MHz. The giso of 2.06 also agrees well with the value derived from the magneticsusceptibility measurements. Most importantly, the model and simulationwith two equivalent Cu ions reproduce best both the position and breadthof the g1(A1) features. Attempts to reproduce both the X-and Q-band EPR spectraby simulation with only a single Cu hyperfine interaction were unsuccessfuland unable to reproduce the multifrequency EPR data . Therefore, the multifrequency EPR analysis evidencesthat the unpaired electron in 1-hole 3 is delocalizedover two equivalent Cu ions.The cnature S14. The Z° site, which is the active intermediate speciesduring the N2O catalytic cycle.23 In addition, the maximum Cu hyperfine coupling in 3 (128 MHz) is comparable to that in CuZ° (118 MHz),indicating that the spin population on the Cu ions in these two speciesis similar (4(μ4-S)} sites or model complex D (Z* site) or equally over three(for the reduced CuZ site) or four Cu ions (for model complex D).It is worth noting that spin delocalization equallyover two oppositelypositioned Cu ions has also been observed for the biological 1-holeCu similar 1. In conomplex D 1 show a 4(μ4-S)} clusters (2 and 3-BF4) was further investigatedby density functional theory(DFT)calculations at the B3LYP/def2-TZVP level. The seesaw and square planar{Cu4(μ4-S)} core structures of the cationsof 2 and 3-BF4,respectively, as well as relevant bond lengths/angles, were well reproducedin the optimized structures (τ4(S) = 0.44 and 0.10; Table S3). The singly occupied molecular orbital 328) of 3-BF4 is primarily composed of Cu 3dyz /3dxz  orbitals and the μ4-S 3pz (39%) orbital featuring π-antibondinginteractions . Such π*interactions in the ground state of 3-BF4 contrast the situation in the 1-hole CuZ* site18 and model complex D(2NHCCuS} planes (centered on Cu2 and Cu4) are almostperpendicular to the Cu4 base plane (vide supra). Such a configuration, combined with the large Cu–(μ4-S)–Cu angles, enables the effective π/πoverlap of Cu 3dyz/3dxz orbitals and the μ4-S 3pz orbital in 2/3-BF4 . Removing one electronfrom the π* HOMO of 2 upon oxidation strengthensthe Cu–S bonds, especially the Cu2–S1 and Cu4–S1bonds, which accordingly leads to the planarization of the {Cu4(μ4-S)} core structure. The Loewdin spindensity was found to be delocalized over the four Cu ions via theseπ* interactions between the Cu and S atoms, constituting anexcellent superexchange pathway (4-S center (42%) and moderately locatedon the Cu2 and Cu4 ions (each 18%), while the Cu1 and Cu3 ions featureonly minor spin populations (each 5%) (4-S in 3-BF4 (42%) is obviously larger than that in the CuZ*site (14%)17 and model complex D (32%),41 indicating a higher extent ofS participation in the redox process. The large spin delocalizationon the μ4-S and high covalency of the Cu–Sand Cu−NHC bonds might also account for the rather small anisotropyof the EPR spectrum that adopts a small g1 value.57The electronic structureof these {Cucomplex D,42 where2Cu4(μ4-S)]3+/2+ redox couple, Cu K-edge X-ray absorption (XAS), and Cu and S valenceto core (VtC) X-ray emission spectra (XES) of 2 and 3 were measured. These data, together with the correspondingcalculated spectra, are shown in Figures S34–S36. The Cu K-edge XAS  clearlyevidences an increase in the rising edge position of 3 as compared to that of 2 (by ∼1 eV) consistentwith a significant metal-based contribution to the oxidation eventin 3. The Cu and S VtC XES probe the transitions fromthe filled valence levels to the 1s core holes on the Cu and S, respectively;59 hence, taken together, the relative contributions of copper andsulfur to the filled bonding levels can be experimentally assessed.The Cu VtC XES  shows verysimilar spectra for 2 and 3, highlightingthat the Cu K-edge XAS, which primarily probes the lowest unoccupiedmolecular orbital (LUMO), is a more sensitive electronic structureprobe in the present case. It is of interest to note that the S VtCXES  shows clear changes between 2 and 3, suggesting a modulation of the sulfurinvolvement in bonding, which may be attributed to either a substantialligand-based oxidation or increased sulfur covalency. Time-dependent(TD)-density functional theory (DFT) and one-electron DFT calculationswere utilized to calculate the XAS and XES spectra, respectively.The general trends in the data are well-reproduced by the calculations,supporting that the DFT electronic structures of 2 and 3 are reasonable descriptions of the electronic ground states. Complex 2 is best described as a fully reduced cluster, while 3 has a hole character with a LUMO that is ∼50% copper-basedand ∼50% ligand-based, reflecting the high covalency of thecluster .To obtain further insight into theelectronic structure variationsof the I2 . The large steric hindrance aroundthe metal ions enforced by the two macrocyclic ligands may accountfor the inertness of 2 toward these substrates. Thoughthe four Cu ions are shielded by the macrocyclic ligands, however,an open cleft allowing for access to the central μ4-S is present on one side of the Cu4S cluster in the 0-holestate, which could make an attack on the S atom possible; this contraststhe situation in the planar 1-hole state Cu4S cluster,where this cleft is mostly closed . The large contribution of the μ4-S 3pz orbital (39%), which is perpendicular to the Cu4S plane, to the HOMO of 2 was expected to imparta nucleophilic character to the S center. Indeed, 2 reactsrapidly with electrophilic [Me3O]BF4 via S-alkylation, giving the dicopper(I) complex 1 and its thiolato-bridged congener [LCuI2(μ2-SMe)]+ . A distinct N2O activation modewhere the S atom is involved as one binding site has been proposed.42 Attack at the S atom  in 2 is an indication of the potentialability of 2 to functionally mimic the CuZ* site. Modification of the macrocyclic ligand to provide a largerring, increased metal ion access, and more electron-rich Cu ions areundergoing.Given that the fully reduced (0-hole) state andthe 1-hole stateare proposed as active states of Cu4(μ4-S)}cluster found in the active CuZ* or CuZ siteof the metalloenzyme N2O reductase, which mediates thefinal step of bacterial denitrification,can be isolated by using a flexible hexadentate macrocyclic ligandsystem L that provides two binding pockets and NHC ligation for theCu ions. This new {Cu4(μ4-S)} clusteris distinct from the very few reported CuZ*/CuZ models that are based on bidentate {PXP} and {NCN} ligands. [L2Cu4(μ4-S)]2+/3+ couldbe isolated in the fully reduced 0-hole state  and in the singly oxidized 1-hole state (S = 1/2), viz. in both states that have been proposed forthe relevant intermediates of CuZ* in the catalytic N2O reduction cycle. Comprehensive structural and spectroscopiccharacterization of the [L2Cu4(μ4-S)]2+/3+ complexes has provided novel insights into howthe {Cu4(μ4-S)} core structure of CuZ* might adapt to the 1e– oxidation statechange, revealing a planarization upon oxidation that gives rise topronounced π* interactions between Cu 3dyz/3dxz orbitals and the μ4-S 3pz orbital. The unpaired electronspin in the 1-hole state is almost equally located on the μ4-S center and on the Cu ions , with metal contributionspredominantly from two Cu ions located opposite each other in the{Cu4(μ4-S)} core. This is similar to whathas been proposed for the active 1-hole intermediate (CuZ°) of CuZ*, and it is also reflected in a significantcontraction of the corresponding Cu–S bonds upon oxidationof the present {Cu4(μ4-S)} model clusterfrom its 0-hole state to its 1-hole state. Cu K-edge XAS and Cu/SK-edge XES spectroscopies, combined with DFT calculations, corroborateda mixed metal- and sulfur-based hole character with a highly covalentCu4S core of the oxidized species.The presentstudy demonstratesthat a model of the unique {Cu4(μ4-S)} core structures of the CuZ(*) site to be largelyunchanged in both resting and active states, structural constraintsimposed by the protein scaffold were considered by fixing the positionof the distal nitrogen or the Cγ atoms of each histidineligand in the DFT geometry optimizations.24 However, theoretical work also indicatedthat, if all constraints are removed, the original seesaw {Cu4(μ4-S)} cluster rearranges to a trigonalpyramidal geometry with three Cu and S atoms at its base and thatthe protein strain energies are higher for the 1-hole species.20 The latter is reminiscent of our 1-hole complex,which indeed undergoes a structural rearrangement from a seesaw toa square planar core geometry. Though the protein environment wassuggested to be strained, the flipping of some histidine rings61 as well as a conformational switch of the activesite relevant to its function has indeed been observed for these N2OR enzymes63 and has been suggested for theirCuA site by theoretical calculations as well.64 Furthermore, a complex network of hydrogen bondsinvolving the protein environment has been suggested to determinethe orientation of the histidine side chains.65 Therefore, a change in local pH or a shift in hydrogen bonding couldlead to a change in the orientation of the histidine ligands, resultingin a corresponding change in the geometry of the {Cu4(μ4-S)} core. Based on this, geometric and electronic structurevariations of the CuZ(*) sites are conceivable when shuttlingthrough different states during N2O reduction; the presentpair of bioinspired {Cu4(μ4-S)} clusters 2/3 clearly illustrates possible variations inresponse to the 1e– oxidation state change.How can thesefindings be related to the enzyme active site? Inprevious computational models that predicted the {Cu


Rousettus aegyptiacus). Bats were anesthetized with a combination of alfaxalone, midazolam, and morphine administered subcutaneously. Incisional line infiltration using bupivacaine was administered in all bats, and additional bilateral intratesticular injection was administered in the males. Ovariectomy was performed via a dorsal approach, by bilateral midline skin incisions at the paralumbar fossa level. Orchiectomy was performed via a ventral approach, by bilateral midline incisions of scrotal skin above the testes. Following surgery, all bats were administered flumazenil for midazolam reversal, and meloxicam for postoperative analgesia, subcutaneously. All bats recovered from anesthesia uneventfully. Bats were monitored for complications up to 10 days following surgery, when skin sutures were removed. No morbidities or mortalities occurred during this period in any of the bats. In conclusion, ventral approach orchiectomy and dorsal approach ovariectomy using the injectable combination alfaxalone-midazolam-morphine in conjunction with local anesthesia and meloxicam are feasible procedures in Egyptian fruit bats and can be performed with relative ease. However, further studies using these techniques in a larger group of bats should be performed to establish their safety.The purpose of this report is to describe the anesthetic and analgesic management and the surgical procedures of gonadectomy in six  healthy adult Egyptian fruit bats ( Rousettus aegyptiacus (R. aegyptiacus), are a common display species in zoological parks, and due to traits like longevity, robust immune system, echolocation and high brain to body ratio, this species is studied in laboratory settings  agonist. Alfaxalone has a high therapeutic index, short half-life, and minimal cardiovascular effects at clinical dose, thus, it can be used for injured or compromised patients  is a synthetic neurosteroid anesthetic agent acting as a γ-aminobutyric acid receptor A  for ovariectomy and orchiectomy, respectively. Bats were housed at a bat colony at the Zoological Garden of Tel-Aviv University and were brought to KSVM-VTH via bat carriers shortly prior to surgery. Acclimatization was not performed, as it was a short procedure and bats were returned to the bat facility following recovery from anesthesia. Due to lack of an appropriate fine weight scale, bats' body weight was estimated to be 130 g  and 150 g  based on the authors' experience working with this species.Six healthy adult (age unknown) Anesthesia was induced with a combination of 15 mg/kg alfaxalone , 1.5 mg/kg midazolam  and 1.5 mg/kg morphine  combined in an insulin syringe  administered subcutaneously between the shoulder blades while bats were gently restrained covered with a drape. Following injection, bats were monitored every minute for relaxation signs (head relaxation and decreased wing muscle tone), while held under the drape. First effect appeared 4 ± 1  min following injection. Mean time to recumbency without movement  was 7 ± 1 (5–8) min, although 10 min from injection, one female still showed an occasional ears and wings movement. As anesthetic depth was considered light, the anesthetist decided to administer an extra dose of alfaxalone (5 mg/kg) at 11 min after the initial injection. Mobility then ceased within 2 min. During anesthetic induction, four of the six bats displayed muscle twitching, that subsided after several minutes as anesthetic depth increased.Following aseptic preparation, bupivacaine 0.5% solution  diluted in sodium chloride 0.9%  was infiltrated using an insulin syringe  at the planned incision line prior to surgery . In the fR) was measured by observation of thoracic movements. Body temperature was measured using rectal thermometer. Anesthetic depth was considered appropriate in all bats , and no movement or elevation in heart or respiratory rates in response to surgical stimulation occurred. Physiologic variables and anesthetic complications are presented in During the procedure, anesthetic depth and physiologic variables were monitored every 5 min. Isoflurane was available for rescue anesthesia in case anesthetic depth was considered inadequate. Heart rate (HR) and rhythm were monitored using a doppler ultrasonic flow detector  placed and taped on the sternum at the heart level. Respiratory frequency  were administered subcutaneously, and eye lubrication was applied . During surgery, bats were actively warmed with a forced-air warming system .Bats were placed on an insulated operating table and oxygen was supplemented (1 L/min) A dorsal approach was used to prevent post-operative self-mutilation. Bats were placed in ventral recumbency, and the dorsal skin was shaved and aseptically prepared over the thoraco-paralumbar region and draped. A skin incision (1–1.5 cm) was made ~1 cm lateral to the midline, at the paralumbar fossa region, in a cranial to caudal direction . The fatIn two females, moderate non-specific local hemorrhage of the tissues around the ovary occurred. Local pressure was applied using sterile wooden cotton swabs until the bleeding subsided. The muscle layer was sutured using absorbable ligatures  with a simple continuous pattern, following replacement of the fat pad. Simple continuous subcutaneous sutures , and intradermal continuous sutures  were placed. Finally, for extra protection against mutilation, three to four nylon sutures were placed in the skin in a simple interrupted pattern . Mean ovariectomy time  was 31 ± 5 (26−37) min.Scrotal skin was aseptically prepared and draped. The testis was manually held slightly distal to the body in order to prevent its retraction into the abdomen. A 5–8-mm longitudinal ventral scrotal incision was made through the skin, subcutaneous tissue and vaginal tunics, using number 15 scalpel blade. The testis was exteriorized and the epididymis, ductus deferens, testicular artery and vein (pampiniform plexus) identified. The ductus deferens and testicular vessels were clamped using mosquito hemostats, and three circumferential ligatures  were placed 0.5 cm apart around them. The ductus deferens and testicular vessels were dissected distal to the distal ligature, to disconnect the testis and epididymis from the body. Scrotal skin was sutured using nylon ligatures . Orchiectomy time  was 7–10 min, and bleeding was minimal.Two female bats began flickering their ears during placement of the last skin suture (at 49 and 50 min following anesthetics injection). In order to facilitate recovery, at the end of surgery bats were administered 0.3 mg/kg flumazenil  subcutaneously to antagonize the midazolam .Total anesthesia time (from anesthetics injection to antagonist administration) was 59 ± 7 (52–68) min for ovariectomies and 28–37 min for orchiectomies. Recovery time (from flumazenil administration until climbing to the carrier roof) was 30 ± 5 (22−35) min. Recovery time in the present report was longer than reported for dexmedetomidine-based protocols  . This diFor postoperative analgesia, bats were administered a subcutaneous injection of 1 mg/kg meloxicam  at the end of surgery, and then continued with 1 mg/kg oral meloxicam  every 24 h for two more days.Following complete recovery from anesthesia, bats were taken back to the bat facility and housed in dark, temperature and humidity-controlled conditions. Fruits and water were provided in accordance with recommendations for the species. Bats were individually housed in order to prevent picking and tearing the sutures by other bats. Bats underwent veterinary exam daily and skin sutures were removed after 10 days. No morbidities or mortalities occurred during this period in any of the bats.R. aegyptiacus using a surgical approach and anesthesia and analgesia regime, including local anesthesia, not previously reported in this species.The present report describes the feasibility of performing gonadectomy in R. aegyptiacus undergoing gonadectomy. Both protocols provided quicker induction compared with the present report , although, both protocols required isoflurane supplementation during surgery (mean inspired fraction of 0.7%) , which result in personnel exposure , 9. Seveof 0.7%) .R. aegyptiacus by the authors   . Althougyptiacus . This emR. aegyptiacus under alfaxalone-midazolam , was carried out using a ventral midline incision and all bats recovered uneventfully. Surgical time was longer than the time in the current report and lasted 45–56 min . It is possible that due to the pecking nature of fruit bats they are prone to postoperative self-mutilation compared with vampire bats. The potential surgical complications posed when using the ventral ovariectomy approach, make the dorsal approach potentially superior in R. aegyptiacus, as in the present report surgical time was shorter, and complications seemed to be minimal. However, this technique should be tested in a larger group of bats to validate its safety.Ovariectomy using a ventral, lateral or dorsal approach was described in numerous species including equine, canine, feline and rodents –42. Ovar5–56 min . Anotherreported . The maiVentral approach orchiectomy technique as described in the present report, can be easily used in this species. Testes were retrieved with ease from the abdominal and pre-scrotal positions, vessels were ligated in a simple manner and no potential complications during or following surgery were observed.Rousettus lanosus), and orchiectomy with radiosurgery was performed in 125 Jamaican fruit-eating bats (Artibeus jamaicensis), and one Ruwenzori bat, under general anesthesia. Total surgery time for open orchiectomy was much longer than the total surgery time in the present report (26–38 min compared with 7–10 min). Postoperative complication (prolonged recovery) occurred in 1/14 bats. Radiosurgeries were short, lasting 5–7 min, however, major complications occurred; 4.8% morbidity rate , and 1.6% mortality rate (2 bats)  . ComplicR. aegyptiacus males. The authors reported that it was difficult to identify the mesoductus deferens, and postoperatively 27% of the testes appeared to adhere to the scrotal sac for at least 14 months. Otherwise, the technique appeared quick (Another study described vasectomy performed in nine ed quick . Althoug2, and continuous rectal temperature. Postoperative pain evaluation was not performed, and to the authors' knowledge, there are no specific pain scales for bat species. For this reason, meloxicam was administered postoperatively regardless of pain evaluation. Additionally, the inability to accurately weigh the bats is a major limitation of this report. In very little animals where weight estimation is challenging, small errors can lead to large differences in drug doses.Limitations to this case report include the small number of animals, and lacking the ability to monitor blood pressure, hemoglobin oxygen saturation, electrocardiogram, end tidal COR. aegyptiacus bats and can be performed with relative ease. However, further studies comparing this anesthesia regimen with other protocols and assessing the reported surgical procedures in a larger group of bats should be performed to establish their safety.In summary, ventral approach orchiectomy and dorsal approach ovariectomy using the injectable combination alfaxalone-midazolam-morphine in conjunction with local anesthesia and meloxicam are feasible procedures in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This case series was carried out as a standard treatment in accordance with good clinical practice. Written informed consent was obtained for including the animal in this case series.ST and YS-B contributed to the data acquisition and interpretation, drafted and revised the manuscript, and approved the final version to be submitted.


Rib fractures are the most common injuries in chest trauma. Compared with younger patients, elderly patients with rib fracture have a higher incidence of complications and mortality. A retrospective study was conducted to investigate the effect of internal fixation compared with conservative treatment on the outcome of rib fracture in elderly patients.We used a 1:1 propensity score matching method to perform a retrospective analysis of 703 elderly patients with rib fractures treated in the Thoracic Surgery Department of Beijing Jishuitan Hospital between 2013 and 2020. After matching, the length of hospital stay, death, symptom relief and rib fracture healing were compared between the surgery and the control group.p = 0.000). After 9 months of follow-up, the fracture healing rate in the surgery group was significantly higher than that in the control group . The fracture healing time (p = 0.000), improvement in pain score (p = 0.000) and duration of pain medication use (p = 0.000) were also significantly better in the surgery group than in the control group.The study included 121 patients receiving SSRF in the surgery group and 121 patients receiving conservative treatment in the control group. The length of hospital stay in the surgery group was significantly longer than that in the conservative group (11.39d vs. 9.48d, Compared with conservative treatment, surgical treatment can prolong hospital stay to some extent. However, it has the advantages of more rapid healing and lessened pain. For rib fractures in elderly individuals, surgical treatment is a safe and effective option under strict surgical indications and is recommended. At present, there is no complete Chinese database of chest trauma patients, and it is difficult to obtain accurate data on chest trauma. It is conservatively estimated that the number of patients with rib fracture is 1.5 million to 2 million per year  and the control group (435 cases) according to the surgical stabilization of rib fractures. The inclusion criteria were as follows: (1) age ≥60 years and (2) diagnosis of acute rib fracture based on medical history and imaging evidence. The exclusion criteria were as follows: (1) patients lost to follow-up ; (2) patients with severe injuries other than chest, that is, patients with abbreviated injury scale (AIS) score ≥3 for the head and neck, face, abdomen, limbs or body surface .Due to significant differences in the basic characteristics of the two groups after study inclusion , they co2.3.Surgical treatment: The surgical treatment was surgical stabilization of rib fractures (SSRF), performed under general anesthesia, with all patients in supine or decubitus position. preoperative ultrasonography was used to locate the broken end of the rib fracture and select the appropriate incision approaches according to the rib fracture site and the anatomical characteristics of the chest wall. The incision length is usually between 5 cm and 15 cm. The surgical steps were as follows: cutting the skin, separating the muscle layer along the muscle space, exposing the broken ends. The fracture was fixed by placing a new pure titanium U-shaped plate  at the broken end and ensured that there were 2–3 fixation nails on both sides of the broken end . The wouConservative treatment: Conservative treatment included the usage of rib straps or chest plates to immobilize the thorax, oxygen supplementation, sputum atomization, pain reliever, and management of fracture complications. Patients with pulmonary infection were given anti-inflammatory therapy. Closed thoracic drainage was performed for patients with massive hemothorax and/or pneumothorax. Patients with severe lung injury were treated with mechanical ventilation. Long-term oral administration of Imericoxib tablets 0.1GM BID for analgesia.2.4.(1) The admission status of the patient: length of hospital stay, length of ICU stay, and duration of mechanical ventilation; (2) The number of hospital deaths; (3) Delayed symptoms after treatment: fracture healing rate (follow-up for 9 months), fracture healing time, reduction of pain score, and duration of pain medication use. The evaluation of pain relief and the fracture healing index included the pain score and follow-up fracture healing. All patients were evaluated for the pain score on admission and at discharge. The usage and duration of pain medications were followed up after discharge.Fracture healing was followed up after discharge, mainly assessed by imaging examination, physical examination and clinical symptoms. In this study, we selected a CT scan to examine the healing of the rib fracture because we could clearly see the fracture line on the axis of CT . In both2.5.This was a cohort study. Baseline information of the surgery and control groups included patient age, sex, underlying diseases , smoking, number of fractures, flail chest, fracture complications  judged by imaging changes, AIS score, injury severity score (ISS) score and pain score upon admission. With these basic characteristics as predictive variables, the calliper value was set as 0.005, and the ratio was 1:1. We calculated the bias matching score of each case using the logistic regression equation, and cases with similar scores in the two groups were matched. After matching, we compared the observation indices between the 242 patients in the surgery and control groups to analyse differences in the efficacies of these two treatment methods for acute rib fracture in elderly individuals.2.6.X ± SD), and an independent sample t test was used for comparisons between groups. Quantitative data with a nonnormal distribution were analysed as medians and quartiles [M (p25–p75)], and the Mann‒Whitney U-test was used for comparisons between groups. The Wilcoxon signed-rank test was used for comparison of paired quantitative data. Qualitative data were expressed as the frequency, and the χ2 test was used for comparisons between groups. p < 0.05 was considered to be statistically significant.SPSS version 26  was used for data analysis. Quantitative data with a normal distribution are expressed as the mean ± standard deviation (3.p > 0.050). However, they were significantly different in terms of age, sex, diabetes, number of fractures, flail chest, AIS scores for the chest, limbs and body surface, ISS, and admission pain score (p < 0.050). After matching, none of the baseline conditions were significantly different between the two groups (p > 0.050).Of the 242 patients after matching, 98.8% had multiple rib fractures (239 cases), 36.0% had flail chest (87 cases), 52.9% had minor injuries (128 cases), 35.9% had serious injuries (87 cases), and 11.2% had severe injuries (27 cases). According to the visual analogue scale (VAS) score, 88.4% (214) of the patients had severe pain.To reduce the impact of underlying diseases and injury severity as confounding factors on the evaluation of therapeutic efficacy, we performed a 1:1 matching of age, sex, underlying diseases, smoking, fracture severity and complications in the two groups of patients. After matching, there were no significant differences in these basic conditions between the two treatment groups or within a group before treatment . The mat3.1.p > 0.050); however, the length of hospital stay was significantly longer in the surgery group than in the control group (p < 0.050) (p < 0.050), except that women, those ≥75 years old, and those without pleural effusion did not show significant differences (p > 0.050).There was no significant difference in the length of ICU stay or mechanical ventilation duration between the surgery group and the control group (< 0.050) . Table 33.2.p > 0.050)  .3.3.p < 0.050). The fracture healing rate in the surgery group was significantly higher than in the control group (p < 0.050). Additionally, the healing time in the surgery group was significantly shorter than in the control group (p < 0.050).After 9 months of follow-up, the fracture healing rate was 96.67% in the surgery group and 88.89% in the control group. There was a significant difference in the healing rate , 6 and hp = 0.000), and the pain score was lower after surgical treatment. There were significant differences in the improvement of pain score and the duration of pain medication use between the surgery group and the control group (p < 0.050). Pain relief was significantly better in the surgery group than in the control group, and the duration of pain medication use was significantly shorter in the surgery group  . AdditioHowever, as a special population, there is still a lack of specific research on the evaluation of surgical treatment of rib fractures in patients over 60 years old. Rib fracture is the most common chest trauma and is often associated with adverse outcomes such as lung infection, ARDS, respiratory failure, and death , 22. TheBased on the diagnosis and treatment experience of our center and previous literature reports, there are significant differences in the characteristics of trauma between the elderly and young people. The main cause of injury in the elderly is blunt trauma . ElderlyThis study showed that compared with conservative treatment, surgical treatment can extend the length of hospital stay to some extent, but it has advantages in terms of the fracture healing rate, fracture healing time and fracture pain relief.Slobogean GP et al.  conducteWe found that there was no significant difference in the length of ICU stay or mechanical ventilation between the two groups. However, the length of hospitalization was significantly longer in the surgery group than in the control group. We analysed the baseline factors that may have affected the length of hospital stay. Except for the pleural effusion group, the length of stay in the surgery group was significantly longer than in the control group regardless of whether the patients had flail chest, pneumothorax or atelectasis before surgery. This may be related to the poor recovery ability of elderly individuals after surgery. A recent meta-analysis by Choi J  showed tn = 123) and therefore the correlation analysis was not reliable. In our study, the mortality rate decreased significantly after sample matching, indicating that the mortality results are indeed affected by the sample size. Mortality in both groups was very low, especially in the surgery group, which had a lower mortality than the control group, but the difference was not statistically significant.Few datasets on rib fractures in elderly individuals have been available for previous studies. Nevertheless, previous studies have found that the overall mortality for patients with rib fracture ranges from 0.0% to 33.3%, compared with 0.0% to 30% for patients who received SSRF and 0.0% to 57.9% for those who received nonsurgical treatment , 37, 38.Marasco S et al.  found thPatients in the surgery group were significantly better than those in the control group in terms of the improvement in pain score and the duration of pain medication use . This stAs for the cost of treatment, given the advantages of the surgical group, pain improvement with fewer drugs, less ICU and mechanical ventilation need to make this treatment option more cost-effective. But on the other hand, the cost of surgical implants, surgical procedures, anesthesia, occupational therapy services and initial specialized equipment, as well as consumables, may be a major barrier for acceptance of this technique.Based on a large amount of previous clinical experience, our centre has developed indications for the selection of surgery to treat elderly patients with rib fracture. At present, our centre recommends SSRF for elderly patients with flail chest and who have no contraindications to surgery. For non-flail chest patients with two or more rib fractures and obvious displacement of the broken ends in more than half of the fractures, surgery is recommended. For patients with non-flail chest with complications, conservative analgesia and respiratory management should be routinely used. If not effective, patients with early severe pain and well-aligned broken fracture ends without obvious surgical contraindications are recommended for surgical treatment. Patients with severe complications should be treated with positive pressure ventilation by an endotracheal intubation ventilator. For patients with poor conservative treatment, respiratory deterioration and long-term chest floating, if there is no obvious contraindication, physicians should consider SSRF.There are some limitations to this study. The number of patients was greatly reduced after matching, which may have influenced the results. This study was a single-centre study, and the results showed that the length of hospital stay, length of ICU stay, mechanical ventilation duration, and mortality were all lower than in previous studies. As a primary trauma and burn treatment centre located in Beijing, we receive critical chest trauma patients from all over the country, and our institute is a leader in surgical quality, ICU quality and nursing quality in China. Therefore, the results may not be generalisable to other treatment centres. To more objectively evaluate the curative effect of SSRF in China, studies are needed with larger sample sizes and that include more centres.With the development of surgical techniques and the improvement of quality of life, age is no longer an absolute contraindication for surgical treatment. We hope that surgical treatment can provide better options for elderly patients and improve patients' quality of life. Of course, surgery itself is traumatic, especially for the elderly, so the length of hospital stay after surgery will be prolonged, but the mortality rate of patients does not increase, and the long-term recovery of patients also shows a better effect, we hope that through our efforts can provide more excellent treatment methods for the treatment of patients with rib fracture.5.In conclusion, we studied the efficacy of two treatment methods for elderly rib fracture patients using the propensity score matching method. We found that surgical stabilization of rib fractures extended the length of hospital stay compared with conservative treatment, but it had advantages in terms of the fracture healing rate, healing time, and pain relief. For rib fractures in elderly individuals, surgical treatment does not significantly increase mortality when applied under strict surgical indications.


Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by a constant accumulation of lipids in the liver. This hepatic lipotoxicity is associated with a dysregulation of the first step in lipid catabolism, known as beta oxidation, which occurs in the mitochondrial matrix. Eventually, this dysregulation will lead to mitochondrial dysfunction. To evaluate the possible involvement of mitochondrial DNA methylation in this lipid metabolic dysfunction, we investigated the functional metabolic effects of mitochondrial overexpression of CpG (MSssI) and GpC (MCviPI) DNA methyltransferases in relation to gene expression and (mito)epigenetic signatures. Overall, the results show that mitochondrial GpC and, to a lesser extent, CpG methylation increase bile acid metabolic gene expression, inducing the onset of cholestasis through mito-nuclear epigenetic reprogramming. Moreover, both increase the expression of metabolic nuclear receptors and thereby induce basal overactivation of mitochondrial respiration. The latter promotes mitochondrial swelling, favoring lipid accumulation and metabolic-stress-induced mitophagy and autophagy stress responses. In conclusion, both mitochondrial GpC and CpG methylation create a metabolically challenging environment that induces mitochondrial dysfunction, which may contribute to the progression of MASLD. Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing epidemic, which mirrors the increased trend of obesity in Western diet-consuming countries. It has an estimated prevalence of 20–30% in Europe and is the most common cause of chronic liver disease worldwide ,2. MASLDPNPLA3 gene being one of the main genetic risk factors . In. In27]. α, HNF4) ,43 was id stress . Interesd stress . Hepatic bile acid synthesis is the major catabolic mechanism for cholesterol elimination from the body and is strictly regulated. Interestingly, recent studies identified bile acid metabolic dysregulation-induced cholestasis as a key factor in steatohepatitis disease etiology in MASLD ,49,50,51Furthermore, new evidence links disturbed bile acid metabolism to mitochondrial dysfunctions, including changes in respiration, mitochondrial swelling, and a decrease in mitochondrial transmembrane potential ,53,54. IAltogether, these findings suggest that mitochondrial GpC/CpG methylation elicits metabolic stress damage, which needs to be overcome by a fast turnover of dysfunctional mitochondria through the process of mitophagy. Consequently, mitochondrial methylation could represent another risk factor in MASLD, given the mounting body of evidence demonstrating a robust correlation between impaired mitophagy–autophagy and the progression of MASLD ,59,60,61NR5a2 (LRH1)), known to be involved in upregulation of mitochondrial respiration, as a direct consequence of GpC/CpG mtDNA hypermethylation , which reflects the ‘length-to-width ratio), and surface area in Fiji [p-value < 0.05 was considered statistically significant.Cells were seeded in a T25 culture flask in a humidified atmosphere (37 °C and 5% CO in Fiji , as desc in Fiji . Statist