Cytoplasmic Membrane Research Articles

Antibiofilm and antivirulence efficacy of Pleurotus platypus methanolic extract against Staphylococcus aureus through ROS generation and cell membrane disruption

Multi-drug resistance is recognized as a significant worldwide public health concern in the current century. Biofilm formation further exacerbates bacterial resistance to antibacterial medications, host immunological responses, and phagocytosis, resulting in long-lasting chronic illnesses. Investigating natural resources is a very potent approach for developing alternative anti-infective medications to effectively control multi-drug resistant bacterial infections. In this study, a unique mushroom species namely Pleurotus platypus had been discovered from the Terai-Duars region of West Bengal, India. The myco-chemical profiling and preliminary chemical analysis of Pleurotus platypus methanolic extract determined the significant presence of metabolites belonging to several major chemical classes such as flavonoid, alkaloid, triterpenoid, polyphenol, benzoic acids, coumarin, flavone etc. Most intriguingly, the extract possessed effective antibacterial, antibiofilm and antivirulence properties against Staphylococcus aureus and Methicillin resistant Staphylococcus aureus, one of the most notable drug-resistant opportunistic and nosocomial pathogens. Mechanistically, the mushroom extract enhanced the production of Reactive Oxygen Species (ROS) inside the targeted bacteria, causing alterations in membrane potential, damage to the cellular membrane and further release of intracellular DNA, destined to cell death. Moreover, the methanolic extract reported the eradication of pre-existing biofilms from the urinary catheter surface, hinting towards its future application in the related field. To summarize, Pleurotus platypus methanolic extract could be an excellent alternative antibacterial and antibiofilm therapeutic candidate for the effective management of Staphylococcus infections with improved outcome.

In vitro antifungal activity of extracts and alkaloid compounds from Piper arboreum against dermatophytes

Piper is widely distributed in subtropical regions and species of this genus are known for their potent pharmacological activities. Piper arboreum Aubl. is a traditional medicinal plant popularly known as "pau-de-angola", "jaborandi", and chili pepper, demonstrating antifungal, trypanocidal, antibacterial, and antioxidant activities. The leaves of P. arboreum were extracted using Soxhlet and dichloromethane to obtain the extract, which was then fractionated using solvents of different polarities. Samples were analyzed using ultra-high-performance liquid chromatography coupled with mass spectrometry equipped with an electrospray ionization source. Antifungal microdilution assays were performed, and scanning and transmission electron microscopy were used to assess the invasion of the pretreated nail. The minimum inhibitory concentration (MIC) values of the extract and a dichloromethane fraction were, respectively, 62.5 μg/ml and 16.0 μg/ml against Trichophyton rubrum, and 125 μg/ml and 62.5 μg/ml, and 500 μg/ml and 500 μg/ml against T. mentagrophytes, and Microsporum gypseum, respectively. No growth was observed on nail fragments exposed to the extract (at concentrations > 64 µg/ml and then inoculated with spore suspension. Transmission electron microscopy revealed strong inhibition of hyphal growth and an irregular growth pattern following treatment with the extract and the dichloromethane fraction. Results demonstrated the antifungal properties of the P. arboreum extract and its dichloromethane fraction against dermatophytes, with the identification of three different alkaloid compounds. The cytotoxicity was specific towards the fungal cells, and morphological and ultrastructural analyses indicated damage to the cell wall and cytoplasmic membrane as the potential mechanism of action. The leaf material used to generate the extract can be taken from the plant without any detrimental effect thus enabling strategies to be implemented for the exploitation of this species.

Role of sumoylation modification of sirtuin 3 in hypertension-associated

Abstract Objective The present study was designed to investigate whether mitochondrial dysfunction and oxidative stress in HTN-injured cardiomyocytes are associated with SIRT3 SUMOylation and whether SIRT3 deSUMOylation can ameliorate the related cardiomyocyte injury. Design and method The human cardiomyocyte AC16 cell line was used for the study. The SIRT3-K288R cell line was constructed by lentiviral transfection;The expression of SOD2, acetyl-SOD2 (MnSOD), protein acetylation level, inflammatory vesicle NLRP3, etc. in different subgroups were detected by Western Blot, and the roles of SUMOylation modification and SIRT3 deacetylation in HTN-associated cell injury were investigated by flow cytometry. Results (1) Western Blot results showed that compared to the HTN cell injury model, the cardiomyocytes in the SIRT3-K288R intervention group had increased Acetyl- SOD2/SOD2 levels were increased (p < 0.05) and protein acetylation levels were decreased in the SIRT3-K288R intervention group compared with the HTN cell injury model, indicating that SIRT3 increased the deacetylation effect on mitochondria and increased cellular antioxidant stress enzyme activity; (2) Western Blot results showed that compared with the HTN cell injury model, cardiomyocytes in the SIRT3-K288R intervention group NLRP3 and NOX4 levels were significantly reduced, indicating that SIRT3 deSUMOylation could improve the inflammatory response to Ang-II-related cell injury. (3) Flow cytometry results showed that K288R transfection ameliorated the reduction of cellular membrane potential, suggesting that deSUMOylation modification of SIRT3 reduces Ang-II damage to mitochondrial membrane potential. Conclusions In the HTN-associated cell injury model, the removal of SUMOylation modification by viral transfection could improve oxidative stress, protein acetylation levels and associated inflammatory responses in HTN-injured cardiomyocytes, indicating that SIRT3 SUMOylation may play an important mechanism in HTN-associated cell injury, and reducing SIRT3 SUMOylation modification may be a possible direction for future research.

Phytochemical Analysis, Total Phenolic Content, Hemolytic and Anti-hemolytic Activities of Centaurea iberica (Asteraceae)

Several plants contain chemical substances that have various biological activities, but they also probably cause harmful effects on cellular membranes. This work deals with the Centaurea iberica (Iberian starthistle) plant. The objective of this study is to identify the main chemical groups in the methanolic extract 80% of the plant, determine its total phenolic content, evaluate its toxic effect on human erythrocytes, and also assess its activity in the protection of normal and glucose-6-phosphate dehydrogenase (G6PD) enzyme-deficient human erythrocytes membranes against oxidative hemolysis. Phytochemical analysis revealed that C. iberica extract contains several bioactive compounds. The results also showed that the plant extract contained good level of total phenolic compounds. The extract exhibited a low hemolytic effect. It also showed excellent activity in the protection of normal and G6PD-deficient human erythrocytes against oxidative hemolysis. KEYWORDS Erythrocytes, G6PD, Iberian starthistle, methanolic extract, toxic effect, oxidative hemolysis

(p)ppGpp Buffers Cell Division When Membrane Fluidity Decreases in Escherichia coli.

Fluidity is an inherent property of biological membranes and its maintenance (homeoviscous adaptation) is important for optimal functioning of membrane-associated processes. The fluidity of bacterial cytoplasmic membrane increases with temperature or an increase in the proportion of unsaturated fatty acids and vice versa. We found that strains deficient in the synthesis of guanine nucleotide analogs (p)ppGpp and lacking FadR, a transcription factor involved in fatty acid metabolism exhibited a growth defect that was rescued by an increase in growth temperature or unsaturated fatty acid content. The strain lacking (p)ppGpp was sensitive to genetic or chemical perturbations that decrease the proportion of unsaturated fatty acids over saturated fatty acids. Microscopy showed that the growth defect was associated with cell filamentation and lysis and rescued by combined expression of cell division genes ftsQ, ftsA, and ftsZ from plasmid or the gain-of-function ftsA* allele but not over-expression of ftsN. The results implicate (p)ppGpp in positive regulation of cell division during membrane fluidity loss through enhancement of FtsZ proto-ring stability. To our knowledge, this is the first report of a (p)ppGpp-mediated regulation needed for adaptation to membrane fluidity loss in bacteria.

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

Decoding PTEN: from biological functions to signaling pathways in tumors.

The tumor suppressor gene Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), possessing both protein and lipid phosphatase activities, is frequently mutated in various human cancers. PTEN aberrations disrupt critical cellular processes like proliferation, apoptosis, migration, and invasion, thereby promoting tumor growth. In the cells, PTEN localizes to the nucleus, cytoplasm, or cell membrane, and its roles depends on the subcellular localization. PTEN is regulated at the transcriptional, post-transcriptional, and post-translational levels, implying that its functions on the tumors are complex. The relationship between PTEN abnormalities and tumors has garnered significant interest in recent years. PTEN regulates essential cellular processes involved in tumorigenesis. Mutations or deletions in the PTEN gene often correlate with unfavorable prognosis and increased cancer recurrence. Numerous studies suggest that PTEN expression levels in tumors could be a valuable biomarker for cancer diagnosis, treatment, and predicting patient outcomes. This paper provides a comprehensive review of the biological function, regulatory mechanisms, and post-translational modifications of PTEN. Furthermore, this review explores the expression and regulation of PTEN in different tumor types, as well as its interactions with environmental factors in tumorigenesis. This comprehensive analysis aims to deepen our understanding of the signaling pathways between PTEN and cancer.

Biochemical analysis of packing and assembling heptad repeat motifs in the coronavirus spike protein trimer.

During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology.

Hepatitis B virus entry, assembly, and egress.

SUMMARYHepatitis B virus (HBV) is an important human pathogen that chronically infects approximately 250 million people in the world, resulting in ~1 million deaths annually. This virus is a hepatotropic virus and can cause severe liver diseases including cirrhosis and hepatocellular carcinoma. The entry of HBV into hepatocytes is initiated by the interaction of its envelope proteins with its receptors. This is followed by the delivery of the viral nucleocapsid to the nucleus for the release of its genomic DNA and the transcription of viral RNAs. The assembly of the viral capsid particles may then take place in the nucleus or the cytoplasm and may involve cellular membranes. This is followed by the egress of the virus from infected cells. In recent years, significant research progresses had been made toward understanding the entry, the assembly, and the egress of HBV particles. In this review, we discuss the molecular pathways of these processes and compare them with those used by hepatitis delta virus and hepatitis C virus , two other hepatotropic viruses that are also enveloped. The understanding of these processes will help us to understand how HBV replicates and causes diseases, which will help to improve the treatments for HBV patients.

Aescin Inhibits Herpes simplex Virus Type 1 Induced Membrane Fusion.

Infections with Herpes simplex virus can cause severe ocular diseases and encephalitis. The present study aimed to investigate potential inhibitors of fusion between HSV-1 and the cellular membrane of the host cell. Fusion and entry of HSV-1 into the host cell is mimicked by a virus-free eukaryotic cell culture system by co-expression of the HSV-1 glycoproteins gD, gH, gL, and gB in presence of a gD receptor, resulting in excessive membrane fusion and polykaryocyte formation. A microscopic read-out was used for the screening of potential inhibitors, whereas luminometric quantification of cell-cell fusion was used in a reporter fusion assay. HSV-1 gB was tagged at its C-terminus with mCherry to express mCherry-gB in both assay systems for the visualization of the polykaryocyte formation. Reporter protein expression of SEAP was regulated by a Tet-On 3 G system. The saponin mixture aescin was identified as the specific inhibitor (IC50 7.4 µM, CC50 24.3 µM, SI 3.3) of membrane fusion. A plaque reduction assay on Vero cells reduced HSV-1 entry into cells and HSV-1 cell-to-cell spread significantly; 15 µM aescin decreased relative plaque counts to 41%, and 10 µM aescin resulted in a residual plaque size of 11% (HSV-1 17 syn+) and 2% (HSV-1 ANG path). Release of the HSV-1 progeny virus was reduced by one log step in the presence of 15 µM aescin. Virus particle integrity was mainly unaffected. Analytical investigation of aescin by UHPLC-MS revealed aescin IA and -IB and isoaescin IA and -IB as the main compounds with different functional activities. Aescin IA had the lowest IC50, the highest CC50, and an SI of > 4.6.

PAK4 is Required for Meiotic Resumption, Spindle Assembly, and Cortical Migration in Mouse Oocytes During Meiotic Maturation.

Oocyte meiotic errors can cause infertility, miscarriage, and birth defects. Here the role and the underlying mechanism of p21 activated kinase 4 (PAK4) in mouse oocyte meiosis is evaluated. It is found that PAK4 expression and its phosphorylation are detected in high level at germinal vesicle (GV) stage, and gradually decreased after meiotic resumption in oocytes. PAK4 has direct physical interaction with both mitogen-activated protein kinases 1/2 (MEK1/2) and Paxillin, they are colocalized on the spindle structure during metaphases I and II. Phospho-PAK4 is distributed beneath the cytoplasmic membrane and on the chromosomes, and colocalized with the microtubule organizing center (MTOC) proteins, Pericentrin and γ-tubulin, as well as phosphor-MEK1/2 and phosphor-Paxillin on spindle poles. PAK4 inhibition by chemical inhibitor LCH-7749944, specific Pak4 morpholino oligo or the dominant negative mutant Pak4K350, 351M influence the meiotic resumption, spindle assembly and its cortical migration, and associated with the downregulation in the dephosphorylation of cyclin dependent kinase 1 (CDK1) and the levels of Cyclin B1, MEK1/2, Paxillin, g-tubulin, acetylated a-tubulin, Arp3, and Cofilin phosphorylation in oocytes. In sum, PAK4 functions to sustain the rational levels of Cyclin B1, MEK1/2, Paxillin, y-tubulin, acetylated a-tubulin, Arp3, and phosphor-Cofilin in mouse oocytes, thereby promotes the meiotic resumption, spindle assembly, and migration during meiotic maturation.

Integrative Human Genetic and Cellular Analysis of the Pathophysiological Roles of AnxA2 in Alzheimer's Disease.

Alzheimer's disease (AD) is an intractable and progressive neurodegenerative disease. Amyloid beta (Aβ) aggregation is the hallmark of AD. Aβ induces neurotoxicity through a variety of mechanisms, including interacting with membrane receptors to alter downstream signaling, damaging cellular or organelle membranes, interfering with protein degradation and synthesis, and inducing an excessive immune-inflammatory response, all of which lead to neuronal death and other pathological changes associated with AD. In this study, we extracted gene expression profiles from the GSE5281 and GSE97760 microarray datasets in the GEO (Gene Expression Omnibus) database, as well as from the Human Gene Database. We identified differentially expressed genes in the brain tissues of AD patients and healthy persons. Through GO, KEGG, and ROC analyses, annexin A2 (AnxA2) was identified as a putative target gene. Notably, accumulating evidence suggests that intracellular AnxA2 is a key regulator in various biological processes, including endocytosis, transmembrane transport, neuroinflammation, and apoptosis. Thus, we conducted a series of cell biology experiments to explore the biological function of AnxA2 in AD. The results indicate that AnxA2 gene knockdown primarily affects oxidative phosphorylation, cell cycle, AD, protein processing in the endoplasmic reticulum, SNARE interactions in vesicular transport, and autophagy. In SH-SY5Y cells secreting Aβ42, AnxA2 gene knockdown exacerbated Aβ42-induced cytotoxicity, including cell death, intracellular ROS levels, and neuronal senescence, altered cell cycle, and reduced ATP levels, suggesting its critical role in mitochondrial function maintenance. AnxA2 gene knockdown also exacerbated the inhibitory effect of Aβ42 on cell migration. AnxA2 overexpression reduced the inflammatory response induced by Aβ42, while its absence increased pro-inflammatory and decreased anti-inflammatory responses. Furthermore, AnxA2 gene knockdown facilitated apoptosis and decreased autophagy. These results indicated potential pathophysiological roles of AnxA2 in AD.

Mechanistic effects of lipid binding pockets within soluble signaling proteins: Lessons from acyl-CoA-binding and START-domain-containing proteins.

While lipids serve as important energy reserves, metabolites, and cellular constituents in all forms of life, these macromolecules also function as unique carriers of information in plant communication given their diverse chemical structures. The signal transduction process involves a sophisticated interplay between messengers, receptors, signal transducers, and downstream effectors. Over the years, an array of plant signaling proteins have been identified for their crucial roles in perceiving lipid signals. However, the mechanistic effects of lipid binding on protein functions remain largely elusive. Recent literature has presented numerous fascinating models that illustrate the significance of protein-lipid interactions in mediating signaling responses. This review focuses on the category of lipophilic signaling proteins that encompass a hydrophobic binding pocket located outside of cellular membranes and provides an update on the lessons learned from two of these structures, namely the acyl-CoA-binding and START domains. It begins with a brief overview of the latest advances in understanding the functions of the two protein families in plant communication. The second part highlights five functional mechanisms of lipid ligands in concert with their target signaling proteins.

Weaker neuroligin 2 - neurexin 1β interaction tethers membranes and signal synaptogenesis through clustering.

Single-pass transmembrane proteins neuroligin (NL) and neurexin (NRX) constitute a pair of synaptic adhesion molecules (SAMs) that are essential for the formation of functional synapses. Binding affinities vary by ∼ 1000 folds between arrays of NL and NRX subtypes, which contribute to chemical and spatial specificities. Current structures are obtained with truncated extracellular domains of NL and NRX and are limited to the higher-affinity NL1/4-NRX complexes. How NL-NRX interaction leads to functional synapses remains unknown. Here we report structures of full-length NL2 alone, and in complex with NRX1β in several conformations, which has the lowest affinity among major NL-NRX subtypes. We show how conformational flexibilities may help in adapting local membrane geometry, and reveal mechanisms underlying variations in NL-NRX affinities modulation. We further show that, despite lower affinity, NL2-NRX1β interaction alone is capable of tethering different lipid membranes in total reconstitution, and that NL2 and NRX1β cluster at inter-cellular junctions without the need of other synaptic components. In addition, NL2 combines with the master post-synaptic scaffolding protein gephyrin and clusters neurotransmitter receptors at cellular membrane. These findings suggest dual roles of NL2 - NRX1β interaction - both as mechanical tether, and as signaling receptors, to ensure correct spatial and chemical coordination between two cells to generate function synapses.

Molecular Characterization of Ancient Prosaposin-like Proteins from the Protist Dictyostelium discoideum.

To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis. Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.

Characterization and Proteomic Analysis of Geobacter sulfurreducens PCA under Long-Term Electron-Donor Starvation

The natural environment of Geobacter sulfurreducens is oligotrophic and described as limiting in both electron donors and terminal electron acceptors (TEA). In previous studies we examined the effects of long-term TEA (fumarate) limitation, and in this study we examine long-term batch cultures under limiting electron donor. The microorganism survived under long-term electron donor (acetate) starvation, maintaining a stable population of ∼1–2 × 108 cells mL−1 for >650 days. Proteins that varied in abundance with a high level of statistical significance (p < 0.05) for stages between mid-log to survival phase (acetate starved) were identified using iTRAQ based mass spectroscopy. The most highly represented proteins that significantly increased in level in the survival phase cells are generally membrane-associated and are involved in energy metabolism and protein fate. These results document that changes in the outer and cytoplasmic membranes may help G. sulfurreducens survive during starvation through detection and transport of nutrients into the cell. A sizeable portion of the identified proteins with unknown or hypothetical function further suggest that much of the biological process involved in survival have yet to be fully understood. Geobacter sulfurreducens was also able to survive under long-term TEA-starvation conditions with ferric citrate as TEA and maintained a stable population of 1.5–3 × 107 cells mL−1 for >650 days. We also found that survival phase cells from fumarate-limiting conditions were able to quickly resuscitate and reduce metal such as ferric iron as compared to the mid-log phase cells.

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis, an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro, inside macrophages, and in M. tuberculosis-infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

Functional analysis of PagERF021 gene in salt stress tolerance in Populus alba × P.glandulosa.

Poplar trees are crucial for timber and greening, but high levels of salt in the soil have severely limited the yield of poplar. ETS2 repressor factor (ERF) transcription factors play an important role in growth, development, and stress response in eukaryotes. Our study focused on the PagERF021 gene from Populus alba × P. glandulosa, which was significantly upregulated in various tissues under salt stress. Both the tissue-specific expression pattern and β-glucuronidase (GUS) staining of proPagERF021-GUS plants indicated that this gene was predominantly expressed in the roots and stems. The subcellular localization showed that the protein was only localized in the nucleus. The yeast assay demonstrated that this protein had transcriptional activation activity at its C-terminal and could specifically binding to the MYB-core cis-element. The overexpression of PagERF021 gene could scavenge the accumulation of reactive oxygen species and reduce the degree of cellular membrane damage, indicating that this gene enhanced the salt tolerance of poplars. This finding will provide a feasible insight for future research into the regulatory mechanisms of ERF genes in resisting to abiotic stress and the development of new stress-resistant varieties in plants.

Isolated Perianal Langerhans Cell Histiocytosis in a Human Immunodeficiency Virus-Positive Adult: A Rare Incidental finding

Abstract Introduction/Objective Langerhans cell histiocytosis (LCH) is a rare disorder characterized by abnormal proliferation of antigen-presenting cells, primarily Langerhans cells. Although it commonly affects bones, skin, and lungs, involvement of the gastrointestinal tract is rare. Specifically, isolated perianal LCH is exceptionally uncommon, with only a few documented cases reported in the literature. Here, we report a case of incidental perianal LCH in an adult HIV patient, highlighting the exceptional rarity and clinical importance of this occurrence. Methods/Case Report A 56-year-old Caucasian male with HIV underwent surgical resection of a 0.5 x 0.4 x 0.3 cm verrucous lesion on the posterior anal margin. His past medical history included granulomatous lymphadenitis, penile warts, and anal dysplasia. Microscopic examination of the excised tissue revealed Langerhans cells with characteristic features, including abundant eosinophilic cytoplasm and irregular nuclear membranes (resembling “coffee-bean” or “folded paper” appearance). Immunohistochemical analysis confirmed the diagnosis by demonstrating positivity for CD1a, S100, and CD68. The BRAF mutation was evaluated on the same paraffin-embedded block, which revealed the lack of the mutant BRAF V600E. PET-CT imaging showed no evidence of metastatic disease, emphasizing the isolated nature of the perianal LCH in this patient. Results (if a Case Study enter NA) NA Conclusion This case highlights the significance of recognizing and thoroughly evaluating rare and incidental conditions such as LCH in HIV patients. Our findings emphasize the importance of maintaining a high index of suspicion for LCH among healthcare providers and ensuring proper follow-up to rule out systemic involvement. Furthermore, there is a need for future studies to investigate the correlation between LCH and HIV. Given its rarity and diverse clinical presentation in immunocompromised individuals, a multidisciplinary approach, tailored treatment plans, and long-term surveillance with imaging modalities like PET/CT are essential for effectively managing adult-onset isolated LCH.

Targeting the Sodium-Potassium Pump as a Therapeutic Strategy in Acute Myeloid Leukemia.

Tissue-specific differences in the expression of paralog genes, which are not essential in most cell types due to the buffering effect of the partner pair, can make for highly selective gene dependencies. To identify selective paralogous targets for acute myeloid leukemia (AML), we integrated the Cancer Dependency Map with numerous datasets characterizing protein-protein interactions, paralog relationships, and gene expression in cancer models. In this study, we identified ATP1B3 as a context-specific, paralog-related dependency in AML. ATP1B3, the β-subunit of the sodium-potassium pump (Na/K-ATP pump), interacts with the α-subunit ATP1A1 to form an essential complex for maintaining cellular homeostasis and membrane potential in all eukaryotic cells. When ATP1B3's paralog ATP1B1 is poorly expressed, elimination of ATP1B3 leads to the destabilization of the Na/K-ATP pump. ATP1B1 expression is regulated through epigenetic silencing in hematopoietic lineage cells through histone and DNA methylation in the promoter region. Loss of ATP1B3 in AML cells induced cell death in vitro and reduced leukemia burden in vivo, which could be rescued by stabilizing ATP1A1 through overexpression of ATP1B1. Thus, ATP1B3 is a potential therapeutic target for AML and other hematologic malignancies with low expression of ATP1B1. Significance: ATP1B3 is a lethal selective paralog dependency in acute myeloid leukemia that can be eliminated to destabilize the sodium-potassium pump, inducing cell death.