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Reports on Counting on Results, a project funded by the Institute of Museum and Library Services that developed and tested tools to standardize and simplify the collection of outcome data from public library patrons. Discusses results of questionnaires that addressed "Planning for Results" service responses. (Author/LRW)

Summary: Mitochondrial function relies on the coordinated transcription of mitochondrial and nuclear genomes to assemble respiratory chain complexes. Across species, the SIN3 coregulator influences mitochondrial functions, but how its loss impacts mitochondrial homeostasis and metabolism in the context of a whole organism is unknown. Exploring this link is important because SIN3 haploinsufficiency causes intellectual disability/autism syndromes and SIN3 plays a role in tumor biology. Here we show that loss of C. elegans SIN-3 results in transcriptional deregulation of mitochondrial- and nuclear-encoded mitochondrial genes, potentially leading to mito-nuclear imbalance. Consistent with impaired mitochondrial function, sin-3 mutants show extensive mitochondrial fragmentation by transmission electron microscopy (TEM) and in vivo imaging, and altered oxygen consumption. Metabolomic analysis of sin-3 mutant animals revealed a mitochondria stress signature and deregulation of methionine flux, resulting in decreased S-adenosyl methionine (SAM) and increased polyamine levels. Our results identify SIN3 as a key regulator of mitochondrial dynamics and metabolic flux, with important implications for human pathologies.

ABSTRACTQuickly understanding the genomic changes that lead to pathogen emergence is necessary to launch mitigation efforts and reduce harm. In this study, we tracked in real time a 2022 bacterial plant disease outbreak in U.S. geraniums (Pelargonium × hortorum) caused by Xhp2022, a novel lineage of Xanthomonas hortorum. Genomes from 31 Xhp2022 isolates from seven states showed limited chromosomal variation and all contained a single plasmid (p93). Time tree and single nucleotide polymorphism whole-genome analysis estimated that Xhp2022 emerged within the last decade. The phylogenomic analysis determined that p93 resulted from the cointegration of three plasmids (p31, p45, and p66) found sporadically across isolates from previous outbreaks. Although p93 had a 49 kb nucleotide reduction, it retained putative fitness genes, which became predominant in the 2022 outbreak. Overall, we demonstrated, through rapid whole-genome sequencing and analysis, a recent, traceable event of genome reduction for niche adaptation typically observed over millennia in obligate and fastidious pathogens.IMPORTANCEThe geranium industry, valued at $4 million annually, faces an ongoing Xanthomonas hortorum pv. pelargonii (Xhp) pathogen outbreak. To track and describe the outbreak, we compared the genome structure across historical and globally distributed isolates. Our research revealed Xhp population has not had chromosome rearrangements since 1974 and has three distinct plasmids. In 2012, we found all three plasmids in individual Xhp isolates. However, in 2022, the three plasmids co-integrated into one plasmid named p93. p93 retained putative fitness genes but lost extraneous genomic material. Our findings show that the 2022 strain group of the bacterial plant pathogen Xanthomonas hortorum underwent a plasmid reduction. We also observed several Xanthomonas species from different years, hosts, and continents have similar plasmids to p93, possibly due to shared agricultural settings. We noticed parallels between genome efficiency and reduction that we see across millennia with obligate parasites with increased niche specificity.

Objetivo: Analisar as publicações científicas relacionadas à atuação do Enfermeiro na prevenção das toxemias gravídicas.Método: Revisão integrativa da literatura. As buscas das evidências científicas foram realizadas nas bases de dados: Literatura Latino-Americana e do Caribe em Ciências da Saúde, Base de Dados de Enfermagem e Medical Literature Analysis and Retrieval System Online via Biblioteca Virtual da Saúde e Scientific Electronic Library Online, utilizando-se dos termos de busca: Assistência de Enfermagem, Cuidados de Enfermagem, Enfermagem, Pré-eclâmpsia e Eclâmpsia, com auxílio dos operadores booleanos “AND” e “OR”. Resultado: Após a seleção, leitura e filtragem, identificaram-se 10 estudos para análise, que foram agrupados por similaridade semântica e discutidos em categorias temáticas, sendo elas: Atuação do Enfermeiro na prevenção das toxemias gravídicas e Dificuldades encontradas na atuação do Enfermeiro na prevenção das toxemias gravídicas. Conclusão: É notável que com a realização de um acompanhamento pré-natal de qualidade durante toda a gestação é possível detectar de forma precoce as alterações nos sinais clínicos causadas pela toxemias gravídicas e iniciar um tratamento adequado.

RESUMO Objetivo Verificar a aplicabilidade do teste de percepção de fala com figuras em crianças com síndrome de Down. Método Estudo observacional, descritivo e prospectivo, realizado em dois centros fonoaudiológicos, aprovado pelos Comitês de Ética em Pesquisa sob número 82522217.5.0000.5440 e 79510317.8.0000.5257. Participaram 41 crianças com síndrome de Down, de ambos os sexos, com idade entre dois anos e dez anos e 11 meses, as quais foram divididas em três grupos: GI (dois a quatro anos e 11 meses); GII (cinco a sete anos e 11 meses) e GIII (oito a dez anos e 11 meses). Foram realizados os procedimentos de anamnese, meatoscopia, audiometria tonal liminar, teste de limiar de recepção de fala com figuras e imitanciometria. Para a análise estatística, Teste Exato de Fisher com nível de significância de 5%. Resultados Ao ser analisado o número de acertos e erros, em relação à idade cronológica, foi encontrada significância para sete palavras: gelo, faca, vaca, chave, rato, cão e sol. Foi analisado, posteriormente, o desempenho no teste de fala com figuras, dos participantes desse estudo e o desempenho dos participantes do estudo que elaborou e validou este teste. Foi observado que, quando se equiparou a porcentagem de acertos nos dois grupos, as palavras com maior ocorrência de acertos foram: mão, casa e sapo. Conclusão: O teste aplicado nesse estudo proporciona a interpretação do resultado de forma clara e objetiva e independe da produção verbal da criança.

The kinase PAR-4/LKB1 is a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use Caenorhabditis elegans to examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a previously unappreciated function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation, but rather to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which both restricts intestinal specification to the E lineage and ensures its robust differentiation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

The intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption and host defense. Studies on mammalian cultured epithelial cells have uncovered some of the molecular players and physical constraints required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not yet been investigated in detail. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode Caenorhabditis elegans to build a high-resolution and dynamic localization map of known and new brush border markers. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation, but become highly stable once microvilli are built. This new toolbox will be instrumental for understanding the molecular processes of microvilli growth and maintenance in vivo, as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP 2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditis elegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP 2 -binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Intestine function relies on the strong polarity of intestinal epithelial cells and the array of microvilli forming a brush border at their luminal pole. Combining a genetic RNA interference (RNAi) screen with in vivo super-resolution imaging in the Caenorhabditis elegans intestine, we found that the V0 sector of the vacuolar ATPase (V0-ATPase) controls a late apical trafficking step, involving Ras-related protein 11 (RAB-11) + endosomes and the N -ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) synaptosome-associated protein 29 (SNAP-29), and is necessary to maintain the polarized localization of both apical polarity modules and brush border proteins. We show that the V0-ATPase pathway also genetically interacts with glycosphingolipids and clathrin in enterocyte polarity maintenance. Finally, we demonstrate that silencing of the V0-ATPase fully recapitulates the severe structural, polarity and trafficking defects observed in enterocytes from individuals with microvillus inclusion disease (MVID) and use this new in vivo MVID model to follow the dynamics of microvillus inclusions. Thus, we describe a new function for V0-ATPase in apical trafficking and epithelial polarity maintenance and the promising use of the C. elegans intestine as an in vivo model to better understand the molecular mechanisms of rare genetic enteropathies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

How tissues from different developmental origins interact to achieve coordinated morphogenesis at the level of a whole organism is a fundamental question in developmental biology. While biochemical signaling pathways controlling morphogenesis have been extensively studied [1-3], morphogenesis of epithelial tissues can also be directed by mechanotransduction pathways physically linking two tissues [4-8]. C. elegans embryonic elongation requires the coordination of three tissues: muscles, the dorsal and ventral epidermis, and the lateral epidermis. Elongation starts by cell-shape changes driven by actomyosin contractions in the lateral epidermis [9, 10]. At mid-elongation, muscles become connected to the apical surface of the dorsal and ventral epidermis by molecular tendons formed by muscle integrins, extracellular matrix, and C. elegans hemidesmosomes (CeHDs). The mechanical signal generated by the onset of muscle contractions in the antero-posterior axis from mid-elongation is translated into a biochemical pathway controlling the maturation of CeHDs in the dorsal and ventral epidermis [11]. Consistently, mutations affecting muscle contractions or molecular tendons lead to a mid-elongation arrest [12]. Here, we found that the mechanical force generated by muscle contractions and relayed by molecular tendons is transmitted by adherens junctions to lateral epidermal cells, where it establishes a newly identified bipolar planar polarity of the apical PAR module. The planar polarized PAR module is then required for actin planar organization, thus contributing to the determination of the orientation of cell-shape changes and the elongation axis of the whole embryo. This mechanotransduction pathway is therefore essential to coordinate the morphogenesis of three embryonic tissues., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID., Methods: Intestinal organoids from Munc18-2 / Stxbp2 -null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions., Results: Loss of Munc18-2 / Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane., Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.

Background Information: Microvillus inclusion disease (MVID) is a genetic disorder affecting intestinal absorption. It is caused by mutations in MYO5B or syntaxin 3 (STX3) affecting apical membrane trafficking. Morphologically, MVID is characterised by a depletion of apical microvilli and the formation of microvillus inclusions inside the cells, suggesting a loss of polarity. To investigate this hypothesis, we examined the location of essential apical polarity determinants in five MVID patients., Results: We found that the polarity determinants Cdc42, Par6B, PKCζ/ι and the structural proteins ezrin and phospho-ezrin were lost from the apical membrane and accumulated either in the cytoplasm or on the basal side of enterocytes in patients, which suggests an inversion of cell polarity. Moreover, microvilli-like structures were observed at the basal side as per electron microscopy analysis. We next performed Myo5B depletion in three dimensional grown human Caco2 cells forming cysts and found a direct link between the loss of Myo5B and the mislocalisation of the same apical proteins; furthermore, we observed that a majority of cysts displayed an inverted polarity phenotype as seen in some patients. Finally, we found that this loss of polarity was specific for MVID: tissue samples of patients with Myo5B-independent absorption disorders showed normal polarity but we identified Cdc42 as a potentially essential biomarker for trichohepatoenteric syndrome., Conclusion: Our findings indicate that the loss of Myo5B induces a strong loss of enterocyte polarity, potentially leading to polarity inversion., Significance: Our results show that polarity determinants could be useful markers to help establishing a diagnosis in patients. Furthermore, they could be used to characterise other rare intestinal absorption diseases., (© 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Cryo-sectioning procedures, initially developed by Tokuyasu, have been successfully improved for tissues and cultured cells, enabling efficient protein localization on the ultrastructural level. Without a standard procedure applicable to any sample, currently existing protocols must be individually modified for each model organism or asymmetric sample. Here, we describe our method that enables reproducible cryo-sectioning of Caenorhabditis elegans larvae/adults and embryos. We have established a chemical-fixation procedure in which flat embedding considerably simplifies manipulation and lateral orientation of larvae or adults. To bypass the limitations of chemical fixation, we have improved the hybrid cryo-immobilization-rehydration technique and reduced the overall time required to complete this procedure. Using our procedures, precise cryo-sectioning orientation can be combined with good ultrastructural preservation and efficient immuno-electron microscopy protein localization. Also, GFP fluorescence can be efficiently preserved, permitting a direct correlation of the fluorescent signal and its subcellular localization. Although developed for C. elegans samples, our method addresses the challenge of working with small asymmetric samples in general, and thus could be used to improve the efficiency of immuno-electron localization in other model organisms., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis., (© 2015. Published by The Company of Biologists Ltd.)

Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.