Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation.

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.

Atomically-Thin Holey 2D Nanosheets of Defect-Engineered MoN-Mo5 N6 Composites as Effective Hybridization Matrices.

The defect engineering of inorganic solids has received significant attention because of its high efficacy in optimizing energy-related functionalities. Consequently, this approach is effectively leveraged in the present study to synthesize atomically-thin holey 2D nanosheets of a MoN-Mo5 N6 composite. This is achieved by controlled nitridation of assembled MoS2 monolayers, which induced sequential cation/anion migration and a gradual decrease in the Mo valency. Precise control of the interlayer distance of the MoS2 monolayers via assembly with various tetraalkylammonium ions is found to be crucial for synthesizing sub-nanometer-thick holey MoN-Mo5 N6 nanosheets with a tunable anion/cation vacancy content. The holey MoN-Mo5 N6 nanosheets are employed as efficient immobilization matrices for Pt single atoms to achieve high electrocatalytic mass activity, decent durability, and low overpotential for the hydrogen evolution reaction (HER). In situ/ex situ spectroscopy and density functional theory (DFT) calculations reveal that the presence of cation-deficient Mo5 N6 domain is crucial for enhancing the interfacial interactions between the conductive molybdenum nitride substrate and Pt single atoms, leading to enhanced electron injection efficiency and electrochemical stability. The beneficial effects of the Pt-immobilizing holey MoN-Mo5 N6 nanosheets are associated with enhanced electronic coupling, resulting in improvements in HER kinetics and interfacial charge transfer.

Hybrid Acid/Base Electrolytic Cell for Hydrogen Generation and Methanol Conversion Implemented by Bifunctional Ni/MoN Nanorod Electrocatalyst.

Combining the methanol oxidation reaction (MOR) and hydrogen evolution reaction (HER) within an integrated electrolytic system may offer the advantages of enhanced kinetics of the anode, reduced energy consumption, and the production of high-purity hydrogen. Herein, it is reported the construction of Ni─MoN nanorod arrays supported on a nickel foam substrate (Ni─MoN/NF) as a bifunctional electrocatalyst for electrocatalytic hydrogen production and selective methanol oxidation to formate. Remarkably, The optimal Ni─MoN/NF catalyst displays exceptional HER performance with an overpotential of only 49 mV to attain 10 mA cm-2 in acid, and exhibits a high activity for MOR to achieve 100 mA cm-2 at 1.48 V in alkali. A hybrid acid/base electrolytic cell with Ni─MoN/NF electrode as anode and cathode is further developed for an integrated HER-MOR cell, which only requires a voltage of 0.56 V at 10 mA cm-2 , significantly lower than that of the HER-OER system (0.70 V). The density functional theory studies reveal that the incorporation of Ni effectively modulates the electronic structure of MoN, thereby resulting in enhanced catalytic activity. The unique combination of high electrocatalytic activity and excellent stability make the Ni─MoN/NF catalyst a promising candidate for practical applications in electrocatalytic hydrogen production and methanol oxidation.

Rab26 controls secretory granule maturation and breakdown in Drosophila.

At the onset of Drosophila metamorphosis, plenty of secretory glue granules are released from salivary gland cells and the glue is deposited on the ventral side of the forming (pre)pupa to attach it to a dry surface. Prior to this, a poorly understood maturation process takes place during which secretory granules gradually grow via homotypic fusions, and their contents are reorganized. Here we show that the small GTPase Rab26 localizes to immature (smaller, non-acidic) glue granules and its presence prevents vesicle acidification. Rab26 mutation accelerates the maturation, acidification and release of these secretory vesicles as well as the lysosomal breakdown (crinophagy) of residual, non-released glue granules. Strikingly, loss of Mon1, an activator of the late endosomal and lysosomal fusion factor Rab7, results in Rab26 remaining associated even with the large glue granules and a concomitant defect in glue release, similar to the effects of Rab26 overexpression. Our data thus identify Rab26 as a key regulator of secretory vesicle maturation that promotes early steps (vesicle growth) and inhibits later steps (lysosomal transport, acidification, content reorganization, release, and breakdown), which is counteracted by Mon1.

Quantitative Detection of the CP4-EPSPS Protein in Genetically Modified Soybeans Using a Novel Fluorescence Immunoassay Assay.

Protein-based detection methods, enzyme-linked immunosorbent assays (ELISAs) and lateral flow strips, have been widely used for rapid, specific, and sensitive detection of genetically modified organisms (GMOs). However, the traditional ELISA method for the quantitative detection of GMOs has certain limitations. Herein, a quantum dot (QD)-based fluorescence-linked immunosorbent assay was developed using QDs as fluorescent markers for the detection of glyphosate-resistant protein (CP4-EPSPS) in the MON89788 soybean. The end-point fluorescent detection system was carried out using QDs conjugated with a goat anti-rabbit secondary antibody. Compared with the conventional sandwich ELISA method, the newly developed fluorescence-linked immunosorbent assay was highly sensitive and accurate for detecting the CP4-EPSPS protein. The quantified linearity was achieved in the range of 0.05-5% (w/w) for the MON89788 soybean sample. The recovery of protein extracted from mixed MON89788 soybean samples ranged from 87.67% to 116.83%. The limits of detection and limits of quantification were 0.7101 and 2.152 pg/mL, respectively. All of the results indicated that the QD-based fluorescence-linked immunosorbent assay was a highly specific and sensitive method for monitoring the CP4-EPSPS protein in GMOs.

Barley endosomal MONENSIN SENSITIVITY1 is a target of the powdery mildew effector CSEP0162 and plays a role in plant immunity.

Encasements formed around haustoria and biotrophic hyphae as well as hypersensitive reaction (HR) cell death are essential plant immune responses to filamentous pathogens. In this study we examine the components that may contribute to the absence of these responses in susceptible barley attacked by the powdery mildew fungus. We find that the effector CSEP0162 from this pathogen targets plant MONENSIN SENSITIVITY1 (MON1), which is important for the fusion of multivesicular bodies to their target membranes. Overexpression of CSEP0162 and silencing of barley MON1 both inhibit encasement formation. We find that the Arabidopsis ecotype No-0 has resistance to powdery mildew, and that this is partially dependent on MON1. Surprisingly, we find the MON1-dependent resistance in No-0 not only includes an encasement response, but also an effective HR. Similarly, silencing of MON1 in barley also blocks Mla3-mediated HR-based powdery mildew resistance. Our results indicate that MON1 is a vital plant immunity component, and we speculate that the barley powdery mildew fungus introduces the effector CSEP0162 to target MON1 and hence reduce encasement formation and HR.

Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway.

4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2Δhsd4A1) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2Δhsd4A1 mutant, HGMS2Δhsd4A1/Δkstd1, enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2Δhsd4A1/kstd2 and HGMS2Δkstd1/Δhsd4A1/kshA1B1, efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Efficient H2 Evolution Coupled with Anodic Oxidation of Iodide over Defective Carbon-Supported Single-Atom Mo-N4 Electrocatalyst.

We successfully prepared nitrogen-doped defective carbon spheres (Mo-N4/d-C) with a high loading of 0.996 wt % via a designed vapor-deposition process for IOR-based hydrogen generation. The synthesized Mo-N4/d-C catalyst provides a record current density of 10 mA cm-2 at 0.77 V. Further, the Mo-N4/d-C catalyst shows a Tafel slope of 25.58 mV dec-1, exceptional stability over time in acidic media, a higher hydrogen generation rate of 0.1063 mL gcat-1 min-1, a high Faradaic efficiency of 99.8%, and a reduction of the energy consumption up to ∼50% for hydrogen evolution by anodic oxidation reaction of iodide (IOR) compared with the conventional OER-based electrolysis. Computational calculations demonstrate that the Mo-N4/d-C structure plays a vital effect on the activity of iodide oxidation, which is competitive with the Pt catalyst.

Spontaneous cutaneous endometriosis in Mons Pubis: review of literature.

Cutaneous endometriosis is not a very often seen condition and is broadly classified as primary (spontaneous) and secondary. While perineal endometriosis arising in a previous scar has been reported, spontaneous cutaneous endometriosis in perineum is extremely rare and only three cases occurring in mons pubis have been reported in literature. We report a case of 34-year-old lady presented with a swelling in pubic region and associated dull aching pain of 1-year duration with no history of cyclical variation of symptoms. Investigations finally concluded a diagnosis of endometriosis and a Complete excision with clear margins. Clinicians should be aware that a spontaneous endometriosis in the perineum can occur and can have atypical presentation with no increase in size or pain during menstruation.

Ammonium stress increases microautophagic activity while impairing macroautophagic flux in Arabidopsis roots.

Plant responses to NH4+ stress are complex, and multiple mechanisms underlying NH4+ sensitivity and tolerance in plants may be involved. Here, we demonstrate that macro- and microautophagic activities are oppositely affected in plants grown under NH4+ toxicity conditions. When grown under NH4+ stress conditions, macroautophagic activity was impaired in roots. Root cells accumulated autophagosomes in the cytoplasm, but showed less autophagic flux, indicating that late steps of the macroautophagy process are affected under NH4+ stress conditions. Under this scenario, we also found that the CCZ1-MON1 complex, a critical factor for vacuole delivery pathways, functions in the late step of the macroautophagic pathway in Arabidopsis. In contrast, an accumulation of tonoplast-derived vesicles was observed in vacuolar lumens of root cells of NH4+ -stressed plants, suggesting the induction of a microautophagy-like process. In this sense, some SYP22-, but mainly VAMP711-positive vesicles were observed inside vacuole in roots of NH4+ -stressed plants. Consistent with the increased tonoplast degradation and the reduced membrane flow to the vacuole due to the impaired macroautophagic flux, the vacuoles of root cells of NH4+ -stressed plants showed a simplified structure and lower tonoplast content. Taken together, this study presents evidence that postulates late steps of the macroautophagic process as a relevant physiological mechanism underlying the NH4+ sensitivity response in Arabidopsis, and additionally provides insights into the molecular tools for studying microautophagy in plants.

Integrative analysis of CRISPR screening data uncovers new opportunities for optimizing cancer immunotherapy.

BACKGROUND: In recent years, the application of functional genetic immuno-oncology screens has showcased the striking ability to identify potential regulators engaged in tumor-immune interactions. Although these screens have yielded substantial data, few studies have attempted to systematically aggregate and analyze them. METHODS: In this study, a comprehensive data collection of tumor immunity-associated functional screens was performed. Large-scale genomic data sets were exploited to conduct integrative analyses. RESULTS: We identified 105 regulator genes that could mediate resistance or sensitivity to immune cell-induced tumor elimination. Further analysis identified MON2 as a novel immune-oncology target with considerable therapeutic potential. In addition, based on the 105 genes, a signature named CTIS (CRISPR screening-based tumor-intrinsic immune score) for predicting response to immune checkpoint blockade (ICB) and several immunomodulatory agents with the potential to augment the efficacy of ICB were also determined. CONCLUSION: Overall, our findings provide insights into immune oncology and open up novel opportunities for improving the efficacy of current immunotherapy agents.

A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.

Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanine-nucleotide-exchange factor (GEF). Here, we show that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli (encoded by CG8270). Bulli localises to Rab7-positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, we observe that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both the Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role for Bulli in the Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.

Novel MLL/KMT2A-MON2 fusion in a child with therapy-related acute myeloid leukemia after treatment for acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML), which is characterized by the reciprocal t (15;17) (q24; q21) translocation, resulting in PML-RARA gene fusion. Therapy-related AML (t-AML) is a serious complication after cytotoxic and/or radiation therapy in many malignant diseases. In this report, MLL/KMT2A-MON2, with balanced chromosomal translocation t (11;12) (q23; q14), was identified as a novel fusion in a child transformed to t-AML after successful treatment of APL. This study emphasized that clinical monitoring with an integrated laboratory approach is essential for the diagnosis and treatment of t-AML.

Close genetic relationship between central Thai and Mon people in Thailand revealed by autosomal microsatellites.

Central Thailand is home to diverse populations with the central Thai constituting the major group, while the Mon, who migrated from southern Myanmar, are sparsely distributed within the region. A total of 338 individuals of eight central Thai (246 samples) and three Mon populations (92 samples) were newly genotyped. When combined with our previously published Mon data, this provides a total of 139 Mon samples. We found genetic similarity between the central Thai and Mon and weak sub-structuring among Thais from central, northern, and northeastern Thailand. The forensic parameter results show high discrimination values which are appropriate for forensic personal identification and paternity testing in both the central Thai and Mon; the probabilities of excluding paternity are 0.999999112 and 0.999999031, respectively, and the combined discrimination power is 0.9999999999999999999999 in both groups. This regional allelic frequency on forensic microsatellites may serve as a useful reference for further forensic investigations in both Thailand and Myanmar.

Event-specific quantitative polymerase chain reaction methods for detection of double-herbicide-resistant genetically modified corn MON 87419 based on the 3'-junction of the insertion site.

MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3'-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419.

Applicability of plasmid calibrant pMON87712 for quantitative detection of the transgenic soybean MON87712.

The transgenic glyphosate-tolerant soybean MON87712 event was developed by the agrochemical and agricultural biotechnology company Monsanto (USA) and commercialized in 2013. Due to the absence of matrix-based and genomic DNA-positive reference material for MON87712, it is very difficult to detect and monitor this event. In this study, we developed a recombinant 760-bp linearized plasmid, including 150 bp of the soybean endogenous lectin gene and 610 bp of the exogenous BBX32 gene plus its 3' flanking sequence of MON87712 by In-Fusion cloning technology. In addition, a duplex real-time polymerase chain reaction for the detection of MON87712 and the soybean endogenous lectin gene was established. By using this method, we achieved specific and quantitative detection of MON87712 in 45 other kinds of crops, with a detection limit of 10 copies/µl. This method provides a new technical means for the accurate detection of transgenic soybean MON87712, as well as technical support for the supervision of agricultural transgenic organisms.

Prevalence of restless leg syndrome in Mongolian adults: Mon-TimeLine study.

We conducted an epidemiologic study based on survey neuroscience screening for restless leg syndrome in the Mongolian to determine the prevalence of restless leg syndrome. We used baseline data from the Mon-TimeLine study, a multidisciplinary, prospective, population-based cohort study. The total number of participants (n = 1572) ranged from 18 to 65 years of age. We found the prevalence of restless leg syndrome to be 6.6% (n = 103), among which 4.1% (n = 16) were males and 7.3% (n = 87) were females. The peak prevalence age for both genders was 45-55, and we found a positive correlation with body mass index (P < 0.05). Most cases were mild (44.7%) or moderate (35.9%), but 18.4% and 1% of the cases were severe or very severe, respectively. The severe groups showed a positive association with chronic kidney disease, as well as with gastritis. In conclusion, the prevalence of restless leg syndrome in Mongolia approximates that in other countries.

MON2 Guides Wntless Transport to the Golgi through Recycling Endosomes.

Endocytic cargos are transported to recycling endosomes (RE) but how these sorting platforms are generated is not well understood. Here we describe our biochemical and live imaging studies of the conserved MON2-DOPEY complex in RE formation. MON2 mainly co-localized with RE marker RAB4B in peripheral dots and perinuclear region. The peripheral RE approached, interacted with, and separated from sorting nexin 3 (SNX3)-positive early endosomes (EE). Membrane-bound DOPEY2 was recruited to RE dependent upon MON2 expression, and showed binding abilities to kinesin and dynein/dynactin motor proteins. MON2-knockout impaired segregation of RE from EE and led to a decreased tubular recycling endosomal network, whereas RE was accumulated at perinuclear regions in DOPEY2-knockout cells. MON2 depletion also impaired intracellular transferrin receptor recycling, as well as retrograde transport of Wntless during its passage through RE before delivery from EE to the Golgi. Together, these data suggest that the MON2 drives separation of RE from EE and is required for efficient transport of endocytic cargo molecules.Key words: membrane trafficking, MON2, recycling endosomes, Wntless.

Whole-genome and enzymatic analyses of an androstenedione-producing Mycobacterium strain with residual phytosterol-degrading pathways.

Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms ß-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrading pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by five groups of enzymes. These five groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid ß-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identified from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl-4-androstene-3,17-dione (9OH-AD) during ß-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purification. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application.

Analysis of Polynomial Nonlinearity Based on Measures of Nonlinearity Algorithms.

We consider measures of nonlinearity (MoNs) of a polynomial curve in two-dimensions (2D), as previously studied in our Fusion 2010 and 2019 ICCAIS papers. Our previous work calculated curvature measures of nonlinearity (MoNs) using (i) extrinsic curvature, (ii) Bates and Watts parameter-effects curvature, and (iii) direct parameter-effects curvature. In this paper, we have introduced the computation and analysis of a number of new MoNs, including Beale's MoN, Linssen's MoN, Li's MoN, and the MoN of Straka, Duník, and S̆imandl. Our results show that all of the MoNs studied follow the same type of variation as a function of the independent variable and the power of the polynomial. Secondly, theoretical analysis and numerical results show that the logarithm of the mean square error (MSE) is an affine function of the logarithm of the MoN for each type of MoN. This implies that, when the MoN increases, the MSE increases. We have presented an up-to-date review of various MoNs in the context of non-linear parameter estimation and non-linear filtering. The MoNs studied here can be used to compute MoN in non-linear filtering problems.