Mechanistic and biophysical characterization of polymyxin resistance response regulator PmrA in Acinetobacter baumannii.

Introduction: Acinetobacter baumannii PmrAB is a crucial two-component regulatory system (TCS) that plays a vital role in conferring resistance to polymyxin. PmrA, a response regulator belonging to the OmpR/PhoB family, is composed of a C-terminal DNA-binding effector domain and an N-terminal receiver domain. The receiver domain can be phosphorylated by PmrB, a transmembrane sensor histidine kinase that interacts with PmrA. Once phosphorylated, PmrA undergoes a conformational change, resulting in the formation of a symmetric dimer in the receiver domain. This conformational change facilitates the recognition of promoter DNA by the DNA-binding domain of PmrA, leading to the activation of adaptive responses. Methods: X-ray crystallography was carried out to solve the structure of PmrA receiver domain. Electrophoretic mobility shift assay and Isothermal titration calorimetry were recruited to validate the interaction between the recombinant PmrA protein and target DNA. Field-emission scanning electron microscopy (FE-SEM) was employed to characterize the surface morphology of A. baumannii in both the PmrA knockout and mutation strains. Results: The receiver domain of PmrA follows the canonical α5ß5 response regulator assembly, which undergoes dimerization upon phosphorylation and activation. Beryllium trifluoride is utilized as an aspartate phosphorylation mimic in this process. Mutations involved in phosphorylation and dimerization significantly affected the expression of downstream pmrC and naxD genes. This impact resulted in an enhanced cell surface smoothness with fewer modifications, ultimately contributing to a decrease in colistin (polymyxin E) and polymyxin B resistance. Additionally, a conservative direct-repeat DNA PmrA binding sequence TTTAAGNNNNNTTTAAG was identified at the promoter region of the pmrC and naxD gene. These findings provide structural insights into the PmrA receiver domain and reveal the mechanism of polymyxin resistance, suggesting that PmrA could be a potential drug target to reverse polymyxin resistance in Acinetobacter baumannii.

Efficacy of immune checkpoint inhibitors along with chemotherapy in non-small cell lung cancer and the impact on adverse reactions and serum tumor markers.

OBJECTIVE: To examine the efficacy of immune checkpoint inhibitors along with chemotherapy in non-small cell lung cancer (NSCLC) and the effect on adverse reactions and serum tumor markers. METHODS: Data of 112 NSCLC patients admitted to Geriatric respiratory department, Xi'an International Medical Center Hospital from February 2018 to March 2021 were analyzed retrospectively. Among them, 54 patients treated with concurrent chemotherapy were labeled as the control group (CG), and 58 patients treated with PD-1/PD-L1 inhibitors in addition to chemotherapy were the observation group (OG). The two groups were compared in terms of immune function indexes, therapeutic efficacy, incidence of adverse reactions, 1-year survival rate, serum tumor markers before and after treatment, and independent risk factors affecting patients' prognosis. RESULTS: Compared to the CG, the OG exhibited significantly better therapeutic efficacy. The levels of IgG, IgA and IgM 6 months after treatment were significantly higher in both groups than those before treatment, and the elevations in the OG were more evident than those in the CG, and the OG demonstrated markedly lower Recombinant Cytokeratin Fragment Antigen 21-1 (CYFRA21-1), Carcinoembryonic antigen (CEA) and Carbohydrate antigen 125 (CA125) levels after treatment than the CG did. Between the two groups, there was no significant difference identified in the incidence of adverse reactions, but the OG was observed to have much higher 1-year survival rate. The pathological stage, differentiation and treatment regimen were independent risk factors affecting patients' prognosis. CONCLUSION: For NSCLC patients, the adoption of PD-1/PD-L1 inhibitors following chemoradiotherapy shows potential in enhancing clinical efficacy, boosting patients' immune function, and improving long-term survival rates, with premising safety profile.

Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin.

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.

Structure and assembly of type VI secretion system cargo delivery vehicle.

Type VI secretion system is widely used in Gram-negative bacteria for injecting toxic effectors into neighboring prokaryotic or eukaryotic cells. Various effectors can be loaded onto the T6SS delivery tube via its core components: Hcp, VgrG, or PAAR. Here, we report 2.8-Å resolution cryo-EM structure of intact T6SS Hcp5-VgrG-PAAR cargo delivery system and crystal structure of unbound Hcp5 from B. fragilis NCTC 9343. Loading of Hcp5 hexameric ring onto VgrG causes expansion of its inner cavity and external surface, explaining how structural changes could be propagated to regulate co-polymerization and surrounding contractile sheath. High-affinity binding between Hcp and VgrG causes entropically unfavorable structuring of long loops. Furthermore, interactions between VgrG trimer and Hcp hexamer are asymmetric, with three of the six Hcp monomers exhibiting a major loop flip. Our study provides insights into the assembly, loading, and firing of T6SS nanomachine that contributes to bacterial inter-species competition and host interactions.

Mechanistic and structural insights into the bifunctional enzyme PaaY from Acinetobacter baumannii.

PaaY is a thioesterase that enables toxic metabolites to be degraded through the bacterial phenylacetic acid (PA) pathway. The Acinetobacter baumannii gene FQU82_01591 encodes PaaY, which we demonstrate to possess γ-carbonic anhydrase activity in addition to thioesterase activity. The crystal structure of AbPaaY in complex with bicarbonate reveals a homotrimer with a canonical γ-carbonic anhydrase active site. Thioesterase activity assays demonstrate a preference for lauroyl-CoA as a substrate. The AbPaaY trimer structure shows a unique domain-swapped C-termini, which increases the stability of the enzyme in vitro and decreases its susceptibility to proteolysis in vivo. The domain-swapped C-termini impact thioesterase substrate specificity and enzyme efficacy without affecting carbonic anhydrase activity. AbPaaY knockout reduced the growth of Acinetobacter in media containing PA, decreased biofilm formation, and impaired hydrogen peroxide resistance. Collectively, AbPaaY is a bifunctional enzyme that plays a key role in the metabolism, growth, and stress response mechanisms of A. baumannii.

Screening and epitope characterization of diagnostic nanobody against total and activated Bacteroides fragilis toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) can rapidly secrete an enterotoxin termed B. fragilis toxin (BFT), which is thought to be the only recognized virulence factor in ETBF. ETBF can cause acute diarrhea, inflammatory bowel disease (IBD), colorectal cancer, and breast cancer. BFT is divided into three subtypes, BFT1, BFT2, and BFT3. BFT1 is the most widely distributed in human B. fragilis isolates. BFT can be used as a biomarker for predicting the inflammation-cancer transformation of intestine and breast. Nanobodies have the advantages of small structure, complete antigen recognition capacity, rapid selection via phage display technology, and can be massively produced in microbial expression systems. Nanobodies have become a powerful tool for medical diagnosis and treatment. This study focuses on screening and structural characterization of nanobodies targeting full length and active BFT. By constructing prokaryotic expression systems to obtain recombinant BFT1 protein, high purity BFT1 protein was used to immunize alpacas. Phage display technology was used to construct a phage display library. The positive clones were selected by bio-panning, and the isothermal titration calorimetry was used to select high-affinity nanobodies. Then the three-dimensional structures of BFT1:Nb2.82 and BFT1:Nb3.27 were solved by crystal X-ray diffraction. We got two kinds of nanobodies, Nb2.82 targeting the BFT1 prodomain and Nb3.27 recognizing the BFT1 catalytic domain. This study provides a new strategy for the early diagnosis of ETBF and the possibility for BFT as a biomarker for diagnosing diseases.

Optimal modes of mind-body exercise for treating chronic non-specific low back pain: Systematic review and network meta-analysis.

Background: There were limited studies that directly compare the outcomes of various mind-body exercise (MBE) therapies on chronic non-specific low back pain (CNLBP). Objectives: To compare the efficacy of the four most popular MBE modes [Pilates, Yoga, Tai Chi (TC), and Qigong] in clinically CNLBP patients, we conducted a systematic review and network meta-analysis (NMA). Methods: We searched databases for eligible randomized controlled trials (RCTs) (from origin to July 2022). RCTs were eligible if they included adults with CNLBP, and implemented one or more MBE intervention arms using Pilates, yoga, TC, and qigong. In addition, pain intensity and physical function were evaluated using validated questionnaires. Results: NMA was carried out on 36 eligible RCTs involving 3,050 participants. The effect of exercise therapy on pain was in the following rankings: Pilates [Surface under cumulative ranking (SUCRA) = 86.6%], TC (SUCRA = 77.2%), yoga (SUCRA = 67.6%), and qigong (SUCRA = 64.6%). The effect of exercise therapy on function: Pilates (SUCRA = 98.4%), qigong (SUCRA = 61.6%,), TC (SUCRA = 59.5%) and yoga (SUCRA = 59.0%). Conclusion: Our NMA shows that Pilates might be the best MBE therapy for CNLBP in pain intensity and physical function. TC is second only to Pilates in improving pain in patients with CNLBP and has the value of promotion. In the future, we need more high-quality, long-term follow-up RCTs to confirm our findings. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=306905, identifier: CRD42022306905.

Is transcranial direct current stimulation beneficial for treating pain, depression, and anxiety symptoms in patients with chronic pain? A systematic review and meta-analysis.

Background: Chronic pain is often accompanied by emotional dysfunction. Transcranial direct current stimulation (tDCS) has been used for reducing pain, depressive and anxiety symptoms in chronic pain patients, but its therapeutic effect remains unknown. Objectives: To ascertain the treatment effect of tDCS on pain, depression, and anxiety symptoms of patients suffering from chronic pain, and potential factors that modulate the effectiveness of tDCS. Methods: Literature search was performed on PubMed, Embase, Web of Science, and Cochrane Library from inception to July 2022. Randomized controlled trials that reported the effects of tDCS on pain and depression and anxiety symptoms in patients with chronic pain were included. Results: Twenty-two studies were included in this review. Overall pooled results indicated that the use of tDCS can effectively alleviate short-term pain intensity [standard mean difference (SMD): -0.43, 95% confidence interval (CI): -0.75 to -0.12, P = 0.007] and depressive symptoms (SMD: -0.31, 95% CI, -0.47 to -0.14, P < 0.001), middle-term depressive symptoms (SMD: -0.35, 95% CI: -0.58 to -0.11, P = 0.004), long-term depressive symptoms (ES: -0.38, 95% CI: -0.64 to -0.13, P = 0.003) and anxiety symptoms (SMD: -0.26, 95% CI: -0.51 to -0.02, P = 0.03) compared with the control group. Conclusion: tDCS may be an effective short-term treatment for the improvement of pain intensity and concomitant depression and anxiety symptoms in chronic pain patients. Stimulation site, stimulation frequency, and type of chronic pain were significant influence factors for the therapeutic effect of tDCS. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=297693, identifier: CRD42022297693.

Effectiveness of mindfulness-based interventions on empathy: A meta-analysis.

Empathy is essential for human survival and social interaction. Although mindfulness-based interventions (MBIs) have been used to improve empathy in healthy populations, its therapeutic efficacy remains unknown. This study aims to investigate the therapeutic effects of MBIs on empathy in a healthy population and the potential factors affecting the efficacy of MBIs. The literature search focused on PubMed, Embase, Web of Science, Cochrane Library, and CNKI from inception to September 2022. Randomized controlled trials and quasi-experimental studies reporting the effects of using MBIs on empathy in healthy populations were included. A total of 13 studies were included in this review. Results of the meta-analysis showed that MBIs improved empathy (SMD, 0.372, 95% CI, 0.164-0.579, p = 0.001) in the healthy population compared with that in the control group. Moreover, results of the subgroup analysis showed that intervention dose (over 24 h vs. under 24 h), format (online vs. offline), and types (different types) were important factors affecting treatment outcomes. This comprehensive review suggests that MBIs are effective treatment for empathy in healthy population. Future research should markedly focus on large-sample, rigorously designed experiments to explore the long-term effects of MBIs on empathy and to elucidate the underlying mechanisms of MBIs. This study provides a reference for the daily application of MBIs.

Single Domain Antibody application in bacterial infection diagnosis and neutralization.

Increasing antibiotic resistance to bacterial infections causes a serious threat to human health. Efficient detection and treatment strategies are the keys to preventing and reducing bacterial infections. Due to the high affinity and antigen specificity, antibodies have become an important tool for diagnosis and treatment of various human diseases. In addition to conventional antibodies, a unique class of "heavy-chain-only" antibodies (HCAbs) were found in the serum of camelids and sharks. HCAbs binds to the antigen through only one variable domain Referred to as VHH (variable domain of the heavy chain of HCAbs). The recombinant format of the VHH is also called single domain antibody (sdAb) or nanobody (Nb). Sharks might also have an ancestor HCAb from where SdAbs or V-NAR might be engineered. Compared with traditional Abs, Nbs have several outstanding properties such as small size, high stability, strong antigen-binding affinity, high solubility and low immunogenicity. Furthermore, they are expressed at low cost in microorganisms and amenable to engineering. These superior properties make Nbs a highly desired alternative to conventional antibodies, which are extensively employed in structural biology, unravelling biochemical mechanisms, molecular imaging, diagnosis and treatment of diseases. In this review, we summarized recent progress of nanobody-based approaches in diagnosis and neutralization of bacterial infection and further discussed the challenges of Nbs in these fields.

A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Progress in structural and functional study of the bacterial phenylacetic acid catabolic pathway, its role in pathogenicity and antibiotic resistance.

Phenylacetic acid (PAA) is a central intermediate metabolite involved in bacterial degradation of aromatic components. The bacterial PAA pathway mainly contains 12 enzymes and a transcriptional regulator, which are involved in biofilm formation and antimicrobial activity. They are present in approximately 16% of the sequenced bacterial genome. In this review, we have summarized the PAA distribution in microbes, recent structural and functional study progress of the enzyme families of the bacterial PAA pathway, and their role in bacterial pathogenicity and antibiotic resistance. The enzymes of the bacterial PAA pathway have shown potential as an antimicrobial drug target for biotechnological applications in metabolic engineering.

Are Mind-Body Exercise Beneficial for Treating Pain, Function, and Quality of Life in Middle-Aged and Old People With Chronic Pain? A Systematic Review and Meta-Analysis.

Background: Aging is a significant risk factor in chronic pain development with extensive disability and greater health care costs. Mind-body exercise (MBE) has been scientifically proven to affect the pain intensity and physical health. Objectives: To assess the effects of MBE modes (Tai Chi, yoga, and qigong) for treating chronic pain among middle-aged and old people, compared with nonactive and active treatment, as well as function, quality of life, and adverse events. Methods: We searched PubMed, Embase, Web of Science, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang Database, and Chinese Scientific Journals Full-Text Database (VIP) till March 2022. No restrictions were chartered within the year and language of publication. We included randomized controlled trials of MBE treatment in middle-aged and elderly people with chronic pain. The overall certainty of evidence was evaluated by using the GRADE approach. Results: A total of 17 studies (n = 1,332) were included in this review. There was low-certainty evidence indicating that MBE had a moderate effect on reducing pain compared with the nonactive and active control group (standard mean difference (SMD): -0.64, 95% confidence interval (CI): -0.86 to -0.42, P < 0.001). Very-low-certainty evidence showed that the pooled SMD for the functional improvement was -0.75 (95% CI: -1.13 to -0.37, P < 0.001). Low-certainty evidence presented that no influence was observed in physical component summary (SMD: 0.23, 95% CI: -0.16 to 0.62, P = 0.24) and mental component summary (SMD: -0.01, 95% CI -0.39 to 0.36, P = 0.95). Conclusion: Our results indicated that MBE was an effective treatment for reducing symptoms of middle-aged and elderly people with chronic pain compared with nonactive and active control groups. TC and qigong had obvious benefits for knee osteoarthritis in self-reported function, but the efficacy of chronic low back pain was uncertain. No significant benefit of MBE on quality of life in older adults with chronic pain was found. More high-quality RCTs should be conducted to explore the efficacy and mechanism of MBE on chronic pain in middle-aged and elderly people from various dimensions, such as affective and cognitive dimensions. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=316591, identifier CRD42022316591.

An Outer Membrane Protein YiaD Contributes to Adaptive Resistance of Meropenem in Acinetobacter baumannii.

Acinetobacter baumannii is an important nosocomial pathogen that can develop various resistance mechanisms to many antibiotics. However, little is known about how it evolves from an antibiotic sensitive to a resistant phenotype. In this study, we investigated the transition of outer membrane proteins (OMPs) under antibiotic stress and identified YiaD as an OMP marker involved in the development of adaptive resistance to meropenem (MEM) in A. baumannii. Following stimulation of a carbapenem-sensitive strain AB5116 with sub-MIC of MEM, yiaD showed significantly decreased expression, and this decrease continued with prolonged stimulation for 8 h. The downregulation of yiaD was not only observed in clinically sensitive strains but also in 45 carbapenem-resistant isolates that produced the ß-lactamases TEM and OXA-23. However, the extent of the reduction of yiaD expression in resistant strains was less than that in sensitive strains. Lack of yiaD resulted in a 4-fold increase in the MIC of AB5116 to MEM. The same level of depressed susceptibility induced by yiaD deletion was observed in both a growth curve test and a survival rate assay. Moreover, the colony shape became enlarged and irregular after loss of yiaD, and the biofilm formation ability of A. baumannii was influenced by YiaD. These results suggest that YiaD could respond to the stimulus of MEM in A. baumannii with a downregulation trend that kept pace with the prolonged stimulation time, indicating that it participates in various routes to benefit MEM resistance evolution in both carbapenem-sensitive and -resistant A. baumannii strains. IMPORTANCE Acinetobacter baumannii can develop various resistance mechanisms to carbapenems. However, the factors involved in the evolutionary process that leads from transition to the sensitive to resistant phenotype are not clear. The outer membrane protein YiaD of A. baumannii was downregulated under the stress of meropenem (MEM), and its expression level was continuously reduced with prolonged stimulation time. The downregulation of yiaD was not only observed in sensitive strains but also in carbapenem-resistant isolates producing the ß-lactamases TEM and OXA-23. However, the extent of yiaD reduction was less in resistant strains than in sensitive strains. Lack of yiaD resulted in an increased MEM MIC, enlarged and irregular colonies, and decreased biofilm formation ability. These results suggest that YiaD responds to MEM stimulus in A. baumannii and participates in the adaptive resistance of MEM in both carbapenem-sensitive and -resistant strains.

Prostate-specific oncogene OTUD6A promotes prostatic tumorigenesis via deubiquitinating and stabilizing c-Myc.

MYC drives the tumorigenesis of human cancers, including prostate cancer (PrCa), thus deubiquitinase (DUB) that maintains high level of c-Myc oncoprotein is a rational therapeutic target. Several ubiquitin-specific protease (USP) family members of DUB have been reported to deubiquitinate c-Myc, but none of them is the physiological DUB for c-Myc in PrCa. By screening all the DUBs, here we reveal that OTUD6A is exclusively amplified and overexpressed in PrCa but not in other cancers, eliciting a prostatic-specific oncogenic role through deubiquitinating and stabilizing c-Myc oncoprotein. Moreover, genetic ablation of OTUD6A efficiently represses prostatic tumorigenesis of both human PrCa cells and the Hi-Myc transgenic PrCa mice, via reversing the metabolic remodeling caused by c-Myc overexpression in PrCa. These results indicate that OTUD6A is a physiological DUB for c-Myc in PrCa setting and specifically promotes prostatic tumorigenesis through stabilizing c-Myc oncoprotein, suggesting that OTUD6A could be a unique therapeutic target for Myc-driven PrCa.

Non-canonical phosphorylation of Bmf by p38 MAPK promotes its apoptotic activity in anoikis.

Bmf contributes to the onset of anoikis by translocating from cytoskeleton to mitochondria when cells lose attachment to the extracellular matrix. However, the structural details of Bmf cytoskeleton tethering and the control of Bmf release upon loss of anchorage remained unknown. Here we showed that cell detachment induced rapid and sustained activation of p38 MAPK in mammary epithelial cell lines. Inhibition of p38 signaling or Bmf knockdown rescued anoikis. Activated p38 MAPK could directly phosphorylate Bmf at multiple sites including a non-proline-directed site threonine 72 (T72). Crystallographic studies revealed that Bmf T72 directly participated in DLC2 binding and its phosphorylation would block Bmf/DLC2 interaction through steric hindrance. Finally, we showed that phosphomimetic mutation of T72 enhanced Bmf apoptotic activity in vitro and in a knock-in mouse model. This work unraveled a novel regulatory mechanism of Bmf activity during anoikis and provided structural basis for Bmf cytoskeleton tethering and dissociation.

A genetically encoded fluorescent biosensor for extracellular L-lactate.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

Photocleavable proteins that undergo fast and efficient dissociation.

Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optogenetic control of biomolecular activities. However, the first generation PhoCl1 exhibited a relatively slow rate of dissociation, potentially limiting its utility. Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.

Proteolysis and multimerization regulate signaling along the two-component regulatory system AdeRS.

Bacterial two-component regulatory systems are ubiquitous environment-sensing signal transducers involved in pathogenesis and antibiotic resistance. The Acinetobacter baumannii two-component regulatory system AdeRS is made up of a sensor histidine kinase AdeS and a cognate response regulator AdeR, which together reduce repression of the multidrug-resistant efflux pump AdeABC. Herein we demonstrate that an N-terminal intrinsically disordered tail in AdeR is important for the upregulation of adeABC expression, although it greatly increases the susceptibility of AdeR to proteasome-mediated degradation. We also show that AdeS assembles into a hexameric state that is necessary for its full histidine kinase activity, which appears to occur via cis autophosphorylation. Taken together, this study demonstrates new structural mechanisms through which two-component systems can transduce environmental signals to impact gene expression and enlightens new potential antimicrobial approach by targeting two-component regulatory systems.