Deletion Mutants Research Articles

A dispensable SepIVA orthologue in Streptomyces venezuelae is associated with polar growth and not cell division

Abstract Background SepIVA has been reported to be an essential septation factor in Mycolicibacterium smegmatis and Mycobacterium tuberculosis. It is a coiled-coil protein with similarity to DivIVA, a protein necessary for polar growth in members of the phylum Actinomycetota. Orthologues of SepIVA are broadly distributed among actinomycetes, including in Streptomyces spp. Results To clarify the role of SepIVA and its potential involvement in cell division in streptomycetes, we generated sepIVA deletion mutants in Streptomyces venezuelae and found that sepIVA is dispensable for growth, cell division and sporulation. Further, mNeonGreen-SepIVA fusion protein did not localize at division septa, and we found no evidence of involvement of SepIVA in cell division. Instead, mNeonGreen-SepIVA was accumulated at the tips of growing vegetative hyphae in ways reminiscent of the apical localization of polarisome components like DivIVA. Bacterial two-hybrid system analyses revealed an interaction between SepIVA and DivIVA. The results indicate that SepIVA is associated with polar growth. However, no phenotypic effects of sepIVA deletion could be detected, and no evidence was observed of redundancy with the other DivIVA-like coiled-coil proteins Scy and FilP that are also associated with apical growth in streptomycetes. Conclusions We conclude that S. venezuelae SepIVA, in contrast to the situation in mycobacteria, is dispensable for growth and viability. The results suggest that it is associated with polar growth rather than septum formation.

Tissue-Specific Effects of Aging on Repeat-Mediated Mutation Hotspots In Vivo

Aging constitutes complex and dynamic alterations in molecular and physiological processes and is associated with numerous disorders, in part due to increased genetic instability. The aging population is projected to double by 2050, underscoring the urgent need to better understand the relationships between aging and age-related disorders. Repetitive DNA elements are intrinsic sources of genetic instability and have been found to co-localize with mutation hotspots in human cancer genomes. In this study, we explored the relationship between aging and DNA repeat-mediated genetic instability in vivo using an H-DNA-forming mirror-repeat sequence from the cancer-associated human c-MYC gene. Utilizing a unique mutation-reporter mouse model, we observed tissue-specific effects of aging on H-DNA-induced genetic instability, with mutation frequencies increasing in spleen tissues and remaining unchanged in testis tissues. Analysis of the mutation spectra revealed large deletion mutations as the primary contributor to increasing H-DNA-induced mutations, supported by increased cleavage activity of H-DNA structures in aged spleen tissues. Our findings demonstrate that aging has distinct tissue-specific effects on repeat-mediated, cancer-associated mutations, providing insights into the complex relationship between aging and cancer.

ARID1A is a Coactivator of STAT5 that Contributes to CD8+ T Cell Dysfunction and Anti-PD-1 Resistance in Gastric Cancer.

ARID1A deletion mutation contributes to improved treatment of several malignancies with immune checkpoint inhibitors (ICIs). However, its role in modulating of tumor immune microenvironment (TIME) of gastric cancer (GC) remains unclear. Here, we report an increase of CD8+ T cells infiltration in GC patients with ARID1A-mutation (MUT), which enhances sensitivity to ICIs. Kaplan-Meier survival analysis showed that ARID1A-mutation patients with gastrointestinal malignancies benefit from immunotherapy. Transcriptome analysis implicated that ARID1A regulates STAT5 downstream targets to inhibit T-cell mediated toxicity. Integrated dual luciferase assay and ChIP-qPCR analyses indicated that ARID1A coordinated with STAT5 to facilitate the transcription of the immunosuppressive factors TGF-β1 and NOX4. ARID1A recruited canonical BAF complex (cBAF) subunits, including SMARCB1 and SMARCD1, to sustain DNA accessibility. Downregulation of ARID1A reduced chromatin remodeling into configurations which make GC more sensitive to ICIs. In addition, targeting STAT5 effectively improved anti-PD-1 efficiency in ARID1A-wild type (WT) GC patients. Taken together, ARID1A is a coactivator of STAT5, function as a chromatin organizer in GC ICIs resistance, and targeting STAT5 is an effective strategy to improve the efficiency of ICIs in GC.

Genome-wide analysis of quorum sensing regulon in marine fish pathogen Vibrio scophthalmi.

Opportunistic fish pathogen Vibrio scophthalmi frequently infects olive flounder and turbot, which are primary marine species cultured for seafood production in Far East Asia. These infections cause substantial yield reductions and significant economic losses. Although quorum sensing (QS) genes were previously reported in V. scophthalmi, the impacts of QS on genome-wide gene expression and consequent behaviors and physiological traits have remained largely unexplored. In this study, we conducted genomic and transcriptomic analyses to uncover the global regulatory network governed by LuxRVs, a QS master regulator in V. scophthalmi. By comparing the wild-type strain and a luxRVs deletion mutant strain, we found that LuxRVs positively regulates biosynthetic genes for poly-hydroxyalkanoate (PHA) while negatively controlling genes for biofilm formation. Quantification of intracellular PHAs and biofilm biomass on borosilicate tubes confirmed these results. Gene set enrichment analyses further demonstrated that LuxRVs also governs genes related to osmoprotection and defense against reactive oxygen species. Overall, these findings indicate that LuxRVs acts as a global transcriptional regulator, controlling a wide range of physiological processes in V. scophthalmi. Targeting LuxRVs could therefore be a promising strategy for improving seafood production by disrupting diverse physiological and pathogenic traits in this fish pathogen.

A new immunomagnetic microfluidic device for characterizing EGFR mutations in circulating tumor cells from patients withnon-small cell lung cancer

Incorporating precision oncology into cancer management has begun to improve clinical outcomes. Accurate sampling techniques that detect molecular aberrations are crucial for effective implementation. Circulating tumor cells (CTCs), derived from primary or metastatic sites and present in the blood, are proposed as useful diagnostic tools, though their use has been limited due to their rarity, especially in early-stage cancers. This study presents a novel immunomagnetic microfluidic device that efficiently isolates CTCs for analyzing epidermal growth factor receptor (EGFR) mutations in patients with non-small cell lung cancer (NSCLC). The device was designed and laser-cut from polymethylmethacrylate. Validation experiments involved spiking PC-9 cells (an established lung cancer cell line containing GLU 746-ALA 750 deletion mutations in exon 19 of the EGFR gene) into media and isolating these cells. Exons 18 – 21 of EGFR were amplified using a polymerase chain reaction to demonstrate the device’s rapid mutation detection capability. Next-generation sequencing was used to characterize these exons in a cohort of 38 NSCLC patients, successfully isolating CTCs from all. Among these patients, 30 (79%) had EGFR mutations, with exon 19 showing the highest mutation rate (87%) and exon 21 the highest point mutation rate (23%). Our device captured CTCs effectively in <1 h, enabling mutation detection. Further studies are needed to assess the prognostic significance of these mutations, but this technology has potential applications in various solid tumors.

SURG-02. ANAPLASTIC PLEOMORPHIC XANTHOASTROCYTOMA WITH LEPTOMENINGEAL DISSEMINATION PRESENTING WITH CRANIAL NEUROPATHY IN AN ADULT PATIENT: ILLUSTRATIVE CASE AND REVIEW OF THE LITERATURE

Abstract INTRODUCTION Anaplastic pleomorphic xanthoastrocytomas (APXA) are very rare, grade III malignant astrocytic glial neoplasms first described as a distinct entity in the 2016 World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS). They are generally seen in pediatric and young adult patients as supratentorial lesions with both solid and cystic components and have a high propensity for recurrence despite local treatment. APXAs occasionally demonstrate leptomeningeal dissemination (LMD) but extremely seldom at the time of diagnosis. They are typically treated via surgical resection with adjuvant chemoradiation, although there is no standard of care for adjuvant therapy. Case Presentation: The authors describe the case of a 37-year-old male with a history of seizures since age 11 who presented with four months of worsening headache, increasingly frequent seizures, and diplopia with right cranial nerve III palsy. Imaging at the time of diagnosis of his seizure disorder as a child demonstrated a small left temporal lesion, which remained stable on imaging for over 20 years and was never biopsied, however MRI at current presentation showed significant interval enlargement with intralesional hemorrhage and leptomeningeal enhancement involving the left temporal and parietal lobes, left internal auditory canal, and right oculomotor nerve. He underwent surgical resection with gross total resection achieved and pathology revealed WHO Grade III pleomorphic xanthoastrocytoma with ATG7::RAF1 fusion, 9p21 deletion, and TERT promoter mutation on next generation sequencing. This was followed by adjuvant whole brain radiation with boost and treatment with MEK inhibitor cobimetinib. His postoperative course was remarkable for intermittent speech difficulty and improving right third nerve palsy. CONCLUSION We describe the unusual presentation of an already exceedingly rare primary CNS tumor, namely APXA in an adult patient with LMD and cranial neuropathy at the time of diagnosis, and detail tailored adjuvant therapy based on genomic profiling.

The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay

3'-Untranslated regions (3'UTRs) are recognized for their role in regulating mRNA turnover while the turnover of a specific group of mRNAs mediated by coding sequences (CDS) remains poorly understood. N4BP1 is a critical inflammatory regulator in vivo with a molecular mechanism that is not yet clearly defined. Our study reveals that N4BP1 efficiently degrades its mRNA targets via CDS rather than the 3'-UTR. This CDS-dependent mRNA turnover mechanism appears to be a general feature of N4BP1, as evidenced by testing multiple mRNA substrates, such as Fos-C, Fos-B, Jun-B and CXCL1. Detailed mapping of the motif identified a crucial 33nt (289-322) sequence near the 5'-end of Fos-C-CDS, where the presence of polyC is necessary for N4BP1-mediated degradation. Functional studies involving domain deletion and point mutations showed that both the KH and NYN domains are essential for N4BP1 to restrict mRNA substrates. The function of N4BP1 in mRNA turnover is not dependent on nonsense-mediated decay as it efficiently restricts mRNA substrates even in cells deficient in UPF1, UPF3A, and UPF3B. Additionally, the function of N4BP1 is not reliant on LUC7L3 despite its known association with this protein. Our findings suggest that N4BP1 acts as an endoribonuclease to degrade mRNA substrates primarily through coding sequences containing a C-rich motif.

Could the transcription factor AtnN coordinating the aspercryptin secondary metabolite gene cluster in Aspergillus nidulans be a global regulator?

Products of dormant secondary metabolite gene clusters of fungal genomes can be exploited for medical purposes as bioactive agents. These clusters can be switched on under oxidative stress and may endow fungi with a versatile chemical armory in a competitive niche. In Aspergillus nidulans, the aspercryptin gene cluster, including the synthase [atnA (AN7884)] and its transcription factor (atnN), was activated under menadione sodium bisulfite (MSB) treatment. In this study, we generated and phenotypically examined the gene deletion and overexpression mutants of atnN and studied the secondary metabolite production of the mutants. Overexpression of atnN significantly reduced the colony growth of surface cultures compared to the control. The ΔatnN gene deletion strain showed higher sensitivity to tert-butyl hydroperoxide (tBOOH), while the atnNOE strain was more resistant to MSB, Congo Red, and sorbitol. Interestingly, deletion of atnN decreased cleistothecia formation of A. nidulans. Manipulation of atnN affected the synthesis of several secondary metabolites, for example, the siderophore production of A. nidulans. The extracellular triacetylfusarinine C (TAFC) production decreased, while the intracellular ferricrocin (FC) concentration of the cultures increased in the atnNOE mutant cultivating A. nidulans in a complex medium containing 1% mycological peptone and 2% maltose. In Czapek-Dox Broth medium, increased asperthecin production was observed in the ΔatnN mutant. The mycotoxin sterigmatocystin synthesis elevated in the ΔatnN mutant, while reduced in the atnNOE mutant on minimal medium. Our study supports previous observations that secondary metabolite production is coordinated in a complex way, and the linkage of stress response, sexual reproduction, and secondary metabolite production can be governed by several transcription factors.

Putative mechanisms of primary resistance to EGFR-targeted therapies: A retrospective study

BackgroundsAdvanced lung adenocarcinoma (LUAD) patient with EGFR mutations often experience resistance to first-line epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) therapy. Nonetheless, the mechanism and biomarkers of primary resistance remain unclear. Further exploration of independent prognostic factors will help clinicians identify patients who may not respond to EGFR-TKIs and select appropriate treatment strategies. MethodsA retrospective study involving 124 patients with stage IV LUAD harboring a common sensitizing EGFR mutation (exon 19 deletion or L858R mutation) who received EGFR-TKIs as first-line therapy was performed. All participants were tested by DNA-targeted sequencing in baseline samples, and there were 19 patients with progression-free survival (PFS) ≤ 3 months (cohort 1, C1, primary resistance), 22 patients with 3 < PFS < 8 months (cohort 2, C2, poor response) without known mutations associated with resistance, and 83 patients with PFS ≥ 8 months (cohort 3, C3, normal). ResultsThe most commonly mutated genes at baseline in patients prior to treatment within the entire study population. were TP53 (65 %), MYC (19 %), CDKN2A (12 %), MUC16 (12 %) and RBM10 (12 %). The baseline characteristics, except for the proportions of patients with EGFR L858R mutation and exon 19 deletion in C1 plus C2 compared to C3 (p = 0.036), were not significantly different among the cohorts. The frequencies of PIK3C2G, STK11, EPAS1, RARA and BTG2 variation were significantly higher in C1, the primary resistance group. Multivariate Cox analysis revealed that PIK3C2G (HR 15.70 95 % CI 3.24–76.05, p < 0.001), STK11 (HR 17.04, 95 % CI 3.68–78.92, p < 0.001), EPAS1 (HR 11.99, 95 % CI 2.57–56.03, p = 0.002), and BTG2 amplification (HR 9.53, 95 % CI 1.67–54.28, p = 0.011) were significantly associated with shorter PFS. ConclusionsThe genomic landscape varies significantly among patients with LUAD, which should be considered when making personalized treatment decisions. This information could provide insights into molecular changes and their effects on clinical treatment in diverse patients with LUAD harboring sensitizing EGFR mutations.

The LysR-type transcriptional factor PacR controls heterocyst differentiation and C/N metabolism in the cyanobacterium Anabaena PCC 7120

PacR (All3953) has previously been identified as a global transcriptional regulator of carbon assimilation in cyanobacteria. In the facultative diazotrophic and filamentous cyanobacterium Anabaena PCC 7120 (Anabaena), inactivation of pacR has been shown to affect cell growth under various conditions. Nitrogen fixation in Anabaena occurs in heterocysts, cells differentiated semiregularly along the filaments following deprivation of combined nitrogen such as nitrate or ammonium. Here, we created a markerless deletion mutant of pacR. In addition to its growth defects observed under different light and nitrogen conditions, the mutant could form a high frequency of heterocysts, including heterocyst doublets, even in the presence of nitrate. Inactivation of pacR led to the upregulation of ntcA, a global regulator of nitrogen metabolism and heterocyst formation, as well as downregulation of genes involved in nitrate uptake and assimilation. These changes led to N-limited cells in the presence of nitrate. PacR also regulates most of the genes encoding bicarbonate transport systems. The promoter regions of ntcA, and several other genes involved in nitrogen or carbon uptake and assimilation, as well as patS and hetN involved in heterocyst patterning can be directly recognized by PacR in vitro. These findings, along with previously reported ChIP-seq data, establish PacR as a crucial transcriptional regulator for balancing carbon and nitrogen metabolism in cyanobacteria.

Osteoprotegerin secretion and its inhibition by RANKL in osteoblastic cells visualized using bioluminescence imaging

Bone remodeling is regulated by the interaction between receptor activator of nuclear factor kappa-B ligand (RANKL) and its receptor RANK on osteoblasts and osteoclasts, respectively. Osteoprotegerin (OPG) is secreted from osteoblasts and inhibits osteoclast differentiation by acting as a decoy receptor for RANKL. Despite its importance, the mechanism underlying the secretion of OPG remains poorly understood. Here, we applied a method of video-rate bioluminescence imaging using a fusion protein with Gaussia luciferase (GLase) and visualized the secretion of OPG from living mouse osteoblastic MC3T3-E1 cells. The bioluminescence imaging revealed that the secretion of OPG fused to GLase (OPG-GLase) occurred frequently and widely across the cell surface. Notably, co-expression of RANKL significantly reduced the secretion of OPG-GLase, indicating an inhibitory role of RANKL on OPG secretion within cells. Further imaging and biochemical analyses using deletion mutants of OPG and RANKL, as well as RANKL mutants that cause autosomal recessive osteopetrosis, demonstrated the essential role of protein-protein interaction between OPG and RANKL in the inhibition of OPG secretion. Treatment with proteasome inhibitors resulted in increased levels of OPG in both culture medium and cell lysates. However, the fold-increase of OPG was similar regardless of the presence or absence of RANKL, suggesting that the regulation of OPG secretion by RANKL is independent of proteasome activity. This report visualized the secretion of OPG from living cells and provided evidence for a novel intracellular inhibitory effect of RANKL on OPG secretion.

Identifying the crucial binding domain of Histain-1 to recombinant TMEM97 in activating chemotactic migration in human corneal epithelial cells.

TMEM97, also known as the sigma-2 receptor, plays a crucial role as an endoplasmic reticular protein involved in various physiological processes such as wound healing, and cholesterol metabolism. Moreover, TMEM97 has been implicated in multiple human diseases including neurodegenerative disorders and cancers. Histatin peptides are endogenous peptides with diverse biological effects, including antimicrobial, immunomodulatory, and wound healing functions. Recent studies have revealed that histatin-1 (Hst1) acts as an endogenous ligand for TMEM97 and is essential for Hst1-induced corneal epithelial migration. In this study, we sought to establish the crucial Hst1 residues that facilitate binding to TMEM97. The purified full-length (FL)-TMEM97 expressed from Escherichia coli exhibited comparable binding affinity, as indicated by the dissociation equilibrium constant (KD) determined by Surface plasmon resonance (SPR), to commercially sourced TMEM97 expressed in mammalian cells. SPR analysis revealed that TMEM97 bound to FL-Hst1 and selected deletion mutants of Hst1. Truncation experiments pinpointed the central region of Hst1 as crucial for its binding to TMEM97, with the loss of residues 15-19 either significantly weakening or completely abolishing the binding interaction. Furthermore, alanine substitution mutant experiments highlighted residues 9-19 as critical for the interaction between TMEM97 and Hst1. Functional assays including migration and signaling were also compared for Hst1 and mutant Hst1. Collectively, these findings underscore the specific binding of Hst1 to TMEM97 and elucidate the critical regions within Hst1 necessary for this interaction which is critically important for the epithelial migration and signaling changes in the ERK and Akt pathways.

Optimized psilocybin production in tryptophan catabolism-repressed fungi.

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an invivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals.

Deciphering FUR-Regulated Gene Networks in Klebsiella pneumoniae Using FUR Knockout mutant

Klebsiella pneumoniae is a nosocomial pathogen causing life-threatening infections worldwide. It possesses many virulence properties and the ferric uptake regulator (FUR) is the transcription factor that regulates the expression of various genes in K. pneumoniae. FUR binds to a conserved 19bp sequence, called the FUR box, for either positive or negative regulation of the gene. FUR alters gene expression according to iron availability and also participates in virulence, colonization & toxin secretion in K. pneumoniae. FUR deletion mutant is an interesting model to understand the virulence factors. In the present study, we identified the genes containing putative FUR boxes (>50% sequence homology) from the genome of K. pneumoniae M33. FUR deletion mutant for K. pneumoniae M33 was created using lambda red recombinase system using the plasmids- pACBSR-hyg, pMDIAI, and pFLP-hyg. The mutant (M33∆FUR) was characterized using various assays like CAS assay, string test, crystal violet assay to access siderophore production, capsule synthesis and biofilm formation respectively. FUR box was present upstream of 18 genes with distinct functions. In the FUR knockout mutant, M33∆FUR an increase in expression of iron transport and siderophore related genes feoC, fhuA, fepB and fes was observed whereas genes feoA, cirA, fecA, fepA and entC were down-regulated. Genes related to biofilm (fimA and mrkD) were downregulated whereas genes related to capsular polysaccharide (rcsA and rcsB) were upregulated upon FUR deletion. Among other genes murG and sucA were downregulated and priB and srlB were upregulated upon FUR deletion. This study shows that FUR regulates many genes involved in virulence, either positively or negatively.

Genetic analysis of a family with hereditary hemorrhagic telangiectasia caused by a novel frameshift deletion mutation of the endoglin (ENG) gene

Genetic analysis of a family with hereditary hemorrhagic telangiectasia caused by a novel frameshift deletion mutation of the endoglin (ENG) gene

Impact of ERG6 Gene Deletion on Membrane Composition and Properties in the Pathogenic Yeast Candida glabrata.

The ERG6 gene is crucial for the biosynthesis of ergosterol, a key component of yeast cell membranes. Our study examines the impact of ERG6 gene deletion on the membrane composition and physicochemical properties of the pathogenic yeast Candida glabrata. Specifically, we investigated changes in selected sterol content, phospholipid composition, transmembrane potential, and PDR16 gene activity. Sterol levels were measured using high-performance liquid chromatography, the phospholipid profile was analysed via thin-layer chromatography, transmembrane potential was assessed with fluorescence spectroscopy, and gene expression levels were determined by quantitative PCR. Our findings revealed a depletion of ergosterol, increased zymosterol and eburicol content, an increased phosphatidylcholine and a reduced phosphatidylethanolamine content in the Δerg6 strain compared to the wt. Additionally, the Δerg6 strain exhibited membrane hyperpolarization without changes in PDR16 expression. Furthermore, the Δerg6 strain showed increased sensitivity to the antifungals myriocin and aureobasidine A. These results suggest that ERG6 gene deletion leads to significant alterations in membrane composition and may activates an alternative ergosterol synthesis pathway in the C. glabrata Δerg6 deletion mutant.

Zinc metalloprotease FgM35, which targets the wheat zinc-binding protein TaZnBP, contributes to the virulence of Fusarium graminearum

Metalloproteinases are ubiquitous in organisms. Most metalloproteinases secreted by pathogenic microorganisms are also called virulence factors, because they degrade proteins in the external tissues of the host, thereby reducing the host’s immunity and increasing its susceptibility to disease. Zinc metalloproteinase is one of the most common metalloproteinases. In our report, we studied the biological function of zinc metalloprotease FgM35 in Fusarium graminearum and the pathogen–host interaction during infection. We found that the asexual and sexual reproduction of the deletion mutant ΔFgM35 were affected, as well as the tolerance of F. graminearum to metal stress. In addition, deletion of FgM35 reduced the virulence of F. graminearum. The wheat target TaZnBP was screened using a wheat yeast cDNA library, and the interaction between FgM35 and TaZnBP was verified by HADDOCK molecular docking, yeast two-hybrid, Bi-FC, Luc, and Co-IP assays. The contribution of TaZnBP to plant immunity was also demonstrated. In summary, our work revealed the indispensable role of FgM35 in the reproductive process and the pathogenicity of F. graminearum, and it identified the interaction between FgM35 and TaZnBP as well as the function of TaZnBP. This provides a theoretical basis for further study of the function of metalloproteinases in pathogen–host interactions.

Sugar alcohol degradation in Archaea: uptake and degradation of mannitol and sorbitol in Haloarcula hispanica

The halophilic archaeon Haloarcula hispanica utilizes the sugar alcohols mannitol and sorbitol as carbon and energy sources. Genes, enzymes, and transcriptional regulators involved in uptake and degradation of these sugar alcohols were identified by growth experiments with deletion mutants and enzyme characterization. It is shown that both mannitol and sorbitol are taken up via a single ABC transporter of the CUT1 transporter family. Then, mannitol and sorbitol are oxidized to fructose by two distinct dehydrogenases. Fructose is further phosphorylated to fructose-1-phosphate by a haloarchaeal ketohexokinase, providing the first evidence for a physiological function of ketohexokinase in prokaryotes. Finally, fructose-1-phosphate is phosphorylated via fructose-1-phosphate kinase to fructose-1,6-bisphosphate, which is cleaved to triosephosphates by a Class I fructose-1,6-bisphosphate aldolase. Two distinct transcriptional regulators, acting as activators, have been identified: an IclR-like regulator involved in activating genes for sugar alcohol uptake and oxidation to fructose, and a GfcR-like regulator that likely activates genes involved in the degradation of fructose to pyruvate. This is the first comprehensive analysis of a sugar alcohol degradation pathway in Archaea.

The Yeast Ribosomal Protein Rpl1b Is Not Required for Respiration.

Previously, Segev and Gerst found that mutants in any of the four ribosomal protein genes rpl1b, rpl2b, rps11a, or rps26b had a petite phenotype-i.e., the mutants were deficient in respiration. Strikingly, mutants of their paralogs rpl1a, rpl2a, rps11b, and rps26a were grande-i.e., competent for respiration. It is remarkable that these paralogs should have opposite phenotypes, because three of the paralog pairs (Rpl1a/Rpl1b, Rpl2a/Rpl2b, Rps11a/Rps11b) are 100% identical to each other in terms of their amino acid sequences, while Rps26a and Rps26b differ in 2 amino acids out of 119. However, while attempting to use this paralog-specific petite phenotype in an unrelated experiment, I found that the rpl1b, rpl2b, rps11a, and rps26b deletion mutants are competent for respiration, contrary to the findings of Segev and Gerst.

CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes.

Focal Segmental Glomerulosclerosis (FSGS) is a histologic lesion caused by a variety of injurious stimuli that lead to dysfunction/loss of glomerular visceral epithelial cells (i.e. podocytes). Pathogenic mutations in CRB2, encoding the type 1 transmembrane protein Crumb 2 Homolog Protein, have been shown to cause early-onset corticosteroid-resistant nephrotic syndrome (SRNS)/FSGS. Here, we identified a 2-generation East Asian kindred (DUK40595) with biopsy-proven SRNS/FSGS caused by a compound heterozygous mutation in CRB2 comprised of the previously described truncating mutation p.Gly1036_Alafs*43 and a rare 9-bp deletion mutation p.Leu1074_Asp1076del. Because compound heterozygous mutations involving the truncating p.Gly1036_Alafs*43 variant have been associated with reduced CRB2 expression in podocytes and autosomal recessive SRNS/FSGS, we sought to define the pathogenic effects of CRB2 deficiency in podocytes. We show that CRB2 knockdown induces YAP activity and target gene expression in podocytes. It upregulates YAP-mediated mechanosignaling and increases the density of focal adhesion and F-actin. Using Elastic Resonator Interference Stress Microscopy (ERISM), we demonstrate that CRB2 knockdown also enhances podocyte contractility in a substrate stiffness-dependent manner. The knockdown effect decreases with increasing substrate stiffness, indicating impaired mechanosensing in CRB2 knockdown cells at low substrate stiffness. While the mechanical activation of CRB2 knockdown cells is associated with increased YAP activity, the enhanced cell contractility is not significantly reduced by the selective YAP inhibitors K-975 and verteporfin, suggesting that multiple pathways may be involved in mechanosignaling downstream of CRB2. Taken together, these studies provide the first evidence that CRB2 deficiency may impair podocyte mechanotransduction via disruption of YAP signaling in podocytes.