Glycyrrhizin, an inhibitor of HMGB1 induces autolysosomal degradation function and inhibits Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori is a key agent for causing gastric complications linked with gastric disorders. In response to infection, host cells stimulate autophagy to maintain cellular homeostasis. However, H. pylori have evolved the ability to usurp the host's autophagic machinery. High mobility group box1 (HMGB1), an alarmin molecule is a regulator of autophagy and its expression is augmented during infection and gastric cancer. Therefore, this study aims to explore the role of glycyrrhizin (a known inhibitor of HMGB1) in autophagy during H. pylori infection. MAIN METHODS: Human gastric cancer (AGS) cells were infected with the H. pylori SS1 strain and further treatment was done with glycyrrhizin. Western blot was used to examine the expression of autophagy proteins. Autophagy and lysosomal activity were monitored by fluorescence assays. A knockdown of HMGB1 was performed to verify the effect of glycyrrhizin. H. pylori infection in in vivo mice model was established and the effect of glycyrrhizin treatment was studied. RESULTS: The autophagy-lysosomal pathway was impaired due to an increase in lysosomal membrane permeabilization during H. pylori infection in AGS cells. Subsequently, glycyrrhizin treatment restored the lysosomal membrane integrity. The recovered lysosomal function enhanced autolysosome formation and concomitantly attenuated the intracellular H. pylori growth by eliminating the pathogenic niche. Additionally, glycyrrhizin treatment inhibited inflammation and improved gastric tissue damage in mice. CONCLUSION: This study showed that inhibiting HMGB1 restored lysosomal activity to ameliorate H. pylori infection. It also demonstrated the potential of glycyrrhizin as an antibacterial agent to address the problem of antimicrobial resistance.

Capsaicin derived from endemic chili landraces combats Shigella pathogen: Insights on intracellular inhibition mechanism.

Ethnic tribals in northeast India have been growing and maintaining local chili landraces for ages. These chilies are known for their characteristic pungency and immense therapeutic properties. Capsaicin, a significant chili metabolite, is recognized as a natural drug for pain relief, diabetic neuropathy, psoriasis, arthritis, etc. In this study, we tried to observe the influence of locality factors on the pungency and bioactive features of Capsicum annuum L. landraces. We also checked the gastro-protective ability of these chilies, especially in the cure of shigellosis. Phytometabolite characterization and estimation were done through spectrophotometric methods. Preparative and analytical HPLC techniques were employed for extracting and purifying capsaicin-enriched fractions. Shigella flexneri growth retardation was determined through the broth dilution method. Gentamicin protection assay and ELISA were done to assess the intracellular invasion and IL-1ß inflammasome production by S.flexneri. The correlation analyses postulated that phenols, flavonoids, chlorophylls, ß-carotene, and capsaicin synthase upregulation strongly influenced capsaicin biosynthesis in chili cultivars. Correspondingly, the inhibitory efficacy of the HPLC-purified Balijuri-derived capsaicin was more effective than the Raja-derived capsaicin in inhibiting intracellular Shigella growth. Reduced levels of pro-inflammatory cytokine (IL1ß) in capsaicin-treated Shigella-infected cells probably reduced inflammation-mediated intestinal damage, limiting bacterial spread. This investigation advocates the unique potential of local chilies in curing deadly 'shigellosis' with mechanistic evidence. Our observation justifies the traditional healing practices of the ethnic people of NE India.

TROPION-Lung08: phase III study of datopotamab deruxtecan plus pembrolizumab as first-line therapy for advanced NSCLC.

Pembrolizumab monotherapy is a standard first-line treatment for PD-L1-high advanced non-small-cell lung cancer (NSCLC) without actionable genomic alterations (AGA). However, few patients experience long-term disease control, highlighting the need for more effective therapies. Datopotamab deruxtecan (Dato-DXd), a novel trophoblast cell-surface antigen 2-directed antibody-drug conjugate, showed encouraging safety and antitumor activity with pembrolizumab in advanced NSCLC. We describe the rationale and design of TROPION-Lung08, a phase III study evaluating safety and efficacy of first-line Dato-DXd plus pembrolizumab versus pembrolizumab monotherapy in patients with advanced/metastatic NSCLC without AGAs and with PD-L1 tumor proportion score ≥50%. Primary end points are progression-free survival and overall survival; secondary end points include objective response rate, duration of response, safety and presence of antidrug antibodies. Clinical trial registration: NCT05215340 (ClinicalTrials.gov).

More than half of patients with non-small-cell lung cancer (NSCLC) are diagnosed when their tumor is advanced (unlikely to be cured with currently available treatments) or metastatic (spread to other parts of the body). These patients have poor survival outcomes. NSCLCs can grow by using a protein called PD-L1 to escape from the immune system. Pembrolizumab is an immunotherapy that targets PD-1, the protein on immune cells that detects PD-L1. Because of this, pembrolizumab prevents the tumor from escaping the immune system by blocking the interaction of PD-L1 with PD-1. Patients whose NSCLC tumors express PD-L1 often respond to pembrolizumab at first but, for most of these patients, their cancer eventually comes back. An investigational drug called datopotamab deruxtecan (Dato-DXd) is a type of therapy called an antibody­drug conjugate that delivers chemotherapy to tumors using an antibody. The antibody in Dato-DXd is directed against a protein called TROP2, which is commonly expressed by tumor cells. Results from early studies show that combining pembrolizumab with Dato-DXd may work well for patients with solid tumors, including NSCLC. This study will look at the benefits and side effects of Dato-DXd added to pembrolizumab compared with pembrolizumab alone as a first treatment option for patients with advanced NSCLC and high levels of PD-L1.

Asiatic acid inhibits intracellular Shigella flexneri growth by inducing antimicrobial peptide gene expression.

AIMS: A rapid rise in resistance to conventional antibiotics for Shigella spp. has created a problem in treating shigellosis. Hence, there is an urgent need for new and non-conventional anti-bacterial agents. The aim of this study is to show how Asiatic acid, a plant-derived compound, inhibits the intracellular growth of Shigella flexneri. METHODS AND RESULTS: Shigella flexneri sensitive and resistant strains were used for checking antimicrobial activity of Asiatic acid by gentamicin protection assay. Asiatic acid inhibited the intracellular growth of all strains. Gene expression analysis showed antimicrobial peptide (AMP) up-regulation by Asiatic acid in intestinal cells. Further western blot analysis showed that ERK, p38, and JNK are activated by Asiatic acid. ELISA was performed to check IL-8, IL-6, and cathelicidin secretion. The antibacterial effect of Asiatic acid was further verified in an in vivo mouse model. CONCLUSIONS: The reason behind the antibacterial activities of Asiatic acid is probably over-expression of antimicrobial peptide genes. Besides, direct antimicrobial activities, antimicrobial peptides also carry immunomodulatory activities. Here, Asiatic acid increased IL-6 and IL-8 secretion to induce inflammation. Overall, Asiatic acid up-regulates antimicrobial peptide gene expression and inhibits intracellular S. flexneri growth. Moreover, Asiatic acid reduced bacterial growth and recovered intestinal tissue damages in in vivo mice model.

Late dominance of the right hemisphere during narrative comprehension.

PET and fMRI studies suggest that auditory narrative comprehension is supported by a bilateral multilobar cortical network. The superior temporal resolution of magnetoencephalography (MEG) makes it an attractive tool to investigate the dynamics of how different neuroanatomic substrates engage during narrative comprehension. Using beta-band power changes as a marker of cortical engagement, we studied MEG responses during an auditory story comprehension task in 31 healthy adults. The protocol consisted of two runs, each interleaving 7 blocks of the story comprehension task with 15 blocks of an auditorily presented math task as a control for phonological processing, working memory, and attention processes. Sources at the cortical surface were estimated with a frequency-resolved beamformer. Beta-band power was estimated in the frequency range of 16-24 Hz over 1-sec epochs starting from 400 msec after stimulus onset until the end of a story or math problem presentation. These power estimates were compared to 1-second epochs of data before the stimulus block onset. The task-related cortical engagement was inferred from beta-band power decrements. Group-level source activations were statistically compared using non-parametric permutation testing. A story-math contrast of beta-band power changes showed greater bilateral cortical engagement within the fusiform gyrus, inferior and middle temporal gyri, parahippocampal gyrus, and left inferior frontal gyrus (IFG) during story comprehension. A math-story contrast of beta power decrements showed greater bilateral but left-lateralized engagement of the middle frontal gyrus and superior parietal lobule. The evolution of cortical engagement during five temporal windows across the presentation of stories showed significant involvement during the first interval of the narrative of bilateral opercular and insular regions as well as the ventral and lateral temporal cortex, extending more posteriorly on the left and medially on the right. Over time, there continued to be sustained right anterior ventral temporal engagement, with increasing involvement of the right anterior parahippocampal gyrus, STG, MTG, posterior superior temporal sulcus, inferior parietal lobule, frontal operculum, and insula, while left hemisphere engagement decreased. Our findings are consistent with prior imaging studies of narrative comprehension, but in addition, they demonstrate increasing right-lateralized engagement over the course of narratives, suggesting an important role for these right-hemispheric regions in semantic integration as well as social and pragmatic inference processing.

Electrophysiological abnormalities as indicators of early-stage pathology in Primary Progressive Aphasia (PPA): A case study in semantic variant PPA.

Language induced and spontaneous oscillatory activity was measured using MEG in a patient with the semantic variant of Primary Progressive Aphasia (svPPA) and 15 healthy controls.The patient showed oscillatory slowing in the left anterior temporal lobe (ATL) that extended into non-atrophied brain tissue in left and right frontal areas. The white matter connections were reduced to the left and right ATL and left frontal regions, exhibiting electrophysiological abnormalities. Altered diffusion metrics in all four language tracts, indicted compromised white matter integrity. Task-related and spontaneous oscillatory abnormalities can indicate early neurodegeneration in svPPA, providing promising targets for intervention.

Brain-Derived Neurotrophic Factor Gene Polymorphism Predicts Response to Continuous Theta Burst Stimulation in Chronic Stroke Patients.

OBJECTIVES: The efficacy of repetitive transcranial magnetic stimulation (rTMS) in clinically relevant neuroplasticity research depends on the degree to which stimulation induces robust, reliable effects. The high degree of interindividual and intraindividual variability observed in response to rTMS protocols, such as continuous theta burst stimulation (cTBS), therefore represents an obstacle to its utilization as treatment for neurological disorders. Brain-derived neurotrophic factor (BDNF) is a protein involved in human synaptic and neural plasticity, and a common polymorphism in the BDNF gene (Val66Met) may influence the capacity for neuroplastic changes that underlie the effects of cTBS and other rTMS protocols. While evidence from healthy individuals suggests that Val66Met polymorphism carriers may show diminished or facilitative effects of rTMS compared to their homozygous Val66Val counterparts, this has yet to be demonstrated in the patient populations where neuromodulatory therapies are most relevant. MATERIALS AND METHODS: We examined the effects of BDNF Val66Met polymorphism on cTBS aftereffects in stroke patients. We compared approximately 30 log-transformed motor-evoked potentials (LnMEPs) obtained per time point: at baseline and at 0, 10, 20, and 30 min after cTBS-600, from 18 patients with chronic stroke using single TMS pulses. We used linear mixed-effects regression with trial-level data nested by subject for higher statistical power. RESULTS: We found a significant interaction between BDNF genotype and pre-/post-cTBS LnMEPs. Val66Val carriers showed decrease in cortical excitability, whereas Val66Met carriers exhibited a modest increase in cortical excitability for 20 min poststimulation, followed by inhibition 30 min after cTBS-600. CONCLUSIONS: Our findings strongly suggest that BDNF genotype differentially affects neuroplastic responses to TMS in individuals with chronic stroke. This provides novel insight into potential sources of variability in cTBS response in patients, which has important implications for optimizing the utility of this neuromodulation approach. Incorporating BDNF polymorphism genetic screening to stratify patients prior to use of cTBS as a neuromodulatory technique in therapy or research may optimize response rates.

Brain-Derived Neurotrophic Factor Polymorphism Influences Response to Single-Pulse Transcranial Magnetic Stimulation at Rest.

OBJECTIVES: The ability of noninvasive brain stimulation to modulate corticospinal excitability and plasticity is influenced by genetic predilections such as the coding for brain-derived neurotrophic factor (BDNF). Otherwise healthy individuals presenting with BDNF Val66Met (Val/Met) polymorphism are less susceptible to changes in excitability in response to repetitive transcranial magnetic stimulation (TMS) and paired associative stimulation paradigms, reflecting reduced neuroplasticity, compared to Val homozygotes (Val/Val). In the current study, we investigated whether BDNF polymorphism influences "baseline" excitability under TMS conditions that are not repetitive or plasticity-inducing. Cross-sectional BDNF levels could predict TMS response more generally because of the ongoing plasticity processes. MATERIALS AND METHODS: Forty-five healthy individuals (23 females; age: 25.3 ± 7.0 years) participated in the study, comprising two groups. Motor evoked potentials (MEP) were collected using single-pulse TMS paradigms at fixed stimulation intensities at 110% of the resting motor threshold in one group, and individually-derived intensities based on MEP sizes of 1 mV in the second group. Functional variant Val66Met (rs6265) was genotyped from saliva samples by a technician blinded to the identity of DNA samples. RESULTS: Twenty-seven participants (60.0%) were identified with Val/Val, sixteen (35.5%) with Val/Met genotype, and two with Met/Met genotype. MEP amplitudes were significantly diminished in the Val/Met than Val/Val individuals. These results held independent of the single-pulse TMS paradigm of choice (p = 0.017110% group; p = 0.035 1 mV group), age, and scalp-to-coil distances. CONCLUSIONS: The findings should be further substantiated in larger-scale studies. If validated, intrinsic differences by BDNF polymorphism status could index response to TMS prior to implementing plasticity-inducing protocols.

Spontaneous oscillatory markers of cognitive status in two forms of dementia.

Abnormal oscillatory brain activity in dementia may indicate incipient neuronal/synaptic dysfunction, rather than frank structural atrophy. Leveraging a potential link between the degree of abnormal oscillatory activity and cognitive symptom severity, one could localize brain regions in a diseased but pre-atrophic state, which may be more amenable to interventions. In the current study, we evaluated the relationships among cognitive deficits, regional volumetric changes, and resting-state magnetoencephalography abnormalities in patients with mild cognitive impairment (MCI; N = 10; age: 75.9 ± 7.3) or primary progressive aphasia (PPA; N = 12; 69.7 ± 8.0), and compared them to normal aging [young (N = 18; 24.6 ± 3.5), older controls (N = 24; 67.2 ± 9.7]. Whole-brain source-level resting-state estimates of relative oscillatory power in the delta (1-4 Hz), theta (4-7 Hz), alpha (8-12 Hz), and beta (15-30 Hz) bands were combined with gray matter volumes and cognitive scores to examine between-group differences and brain-behavior correlations. Language and executive function (EF) abilities were impaired in patients with PPA, while episodic memory was impaired in MCI. Widespread oscillatory speeding and volumetric shrinkage was associated with normal aging, whereas the trajectory in PPA indicated widespread oscillatory slowing with additional volumetric reductions. Increases in delta and decreases in alpha power uniquely predicted group membership to PPA. Beyond volumetric reductions, more delta predicted poorer memory. In patients with MCI, no consistent group difference among oscillatory measures was found. The contributions of delta/alpha power on memory abilities were larger than volumetric differences. Spontaneous oscillatory abnormalities in association with cognitive symptom severity can serve as a marker of neuronal dysfunction in dementia, providing targets for promising treatments.

Structure and dynamics of Helicobacter pylori nickel-chaperone HypA: an integrated approach using NMR spectroscopy, functional assays and computational tools.

Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.

A pediatric institutional acute stroke protocol improves timely access to stroke treatment.

AIM: We aimed to evaluate whether an institutional acute stroke protocol (ASP) could accelerate the diagnosis and secondary treatment of pediatric stroke. METHOD: We initiated an ASP in 2005. We compared 209 children (125 males, 84 females; median age 4.8y, interquartile range [IQR] 1.2-9.3y, range 0.09-17.7y) diagnosed with arterial ischemic stroke 'pre-protocol' (1992-2004) to 112 children (60 males, 52 females; median age 5.8y, IQR 1.0-11.4y, range 0.08-17.7y) diagnosed 'post-protocol' (2005-2012) for time-to-diagnosis, mode of diagnostic imaging, and time-to-treatment with antithrombotic medication (aspirin or anticoagulants). RESULTS: Overall, the interval from symptom onset to diagnosis was similar post-protocol compared to pre-protocol (20.3 vs 22.7h; p=0.109), although mild strokes (Pediatric National Institute of Health Stroke Scale [PedNIHSS] 0-4), were diagnosed faster post-protocol (12.1 vs 36.3h; p=0.003). Magnetic resonance imaging (MRI) was the initial diagnostic modality more often post-protocol (25% vs 1.4%; p<0.001). Initial MRI was more accurate for diagnosing stroke than initial CT (100% vs 47%; p<0.001) with similar time-to-diagnosis. The proportion of children receiving antithrombotic medication within 24 hours doubled in the post-protocol period (83% vs 36%; p<0.001). INTERPRETATION: A pediatric ASP accelerated time-to-treatment, time-to-diagnosis in children with subtle strokes, and increased MRI as initial imaging, reducing the need for computed tomography. Implementing optimized ASPs can facilitate more timely access to diagnosis and management of children with acute stroke.

Clinical management, monitoring, and prophylaxis of adverse events of special interest associated with datopotamab deruxtecan.

Antibody drug conjugates (ADCs) are an emerging class of treatments designed to improve efficacy and decrease toxicity compared with other systemic therapies through the selective delivery of cytotoxic agents to tumor cells. Datopotamab deruxtecan (Dato-DXd) is a novel ADC comprising a topoisomerase I inhibitor payload and a monoclonal antibody directed to trophoblast cell-surface antigen 2 (TROP2), a protein that is broadly expressed in several types of solid tumors. Dato-DXd is being investigated across multiple solid tumor indications. In the ongoing, first-in-human TROPION-PanTumor01 phase I study (ClinicalTrials.gov: NCT03401385), encouraging and durable antitumor activity and a manageable safety profile was demonstrated in patients with advanced/metastatic hormone receptor-positive/human epidermal growth factor receptor2-negative breast cancer (HR+/HER2- BC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC). Improved understanding of the adverse events (AEs) that are associated with Dato-DXd and their optimal management is essential to ensure safe and successful administration. Interstitial lung disease/pneumonitis, infusion-related reactions, oral mucositis/stomatitis, and ocular surface events have been identified as AEs of special interest (AESIs) for which appropriate prevention, monitoring, and management is essential. This article summarizes the incidence of AESIs among patients with HR+/HER2- BC, TNBC, and NSCLC reported in TROPION-PanTumor01. We report our recommendations for AESI prophylaxis, early detection, and management, using experience gained from treating AESIs that occur with Dato-DXd in clinical trials.

Mechanism of copper incorporation in subunit II of cytochrome C oxidase from Thermus thermophilus: identification of intermediate species.

Detailed spectroscopic and kinetic studies of incorporation of copper ion in the wild type (WT) and the D111AA (AA = K, N, or E) mutants of the metal ion binding site of the soluble fragment of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) showed the formation of at least two distinct intermediates. The global analyses of the multiwavelength kinetic results suggested a four-step reaction scheme involving two distinct intermediates in the pathway of incorporation of copper ions into the apoprotein forming the purple dinuclear CuA. An early intermediate similar to the red copper binding proteins was detected in the WT as well as in all the mutants. The second intermediate was a green copper species in the case of WT TtCuA. Mutation of Asp111, however, formed a second intermediate that is distinctly different from that formed in the case of the WT protein, suggesting that mutants follow pathways of copper ion incorporation different from that in the WT protein. The electrostatic interaction between Asp111 and the coordinating His114 possibly plays a subtle role in the mechanism of incorporation of metal ion into the protein. The overall Kd for WT TtCuA was found to be ~8 nM, which changed with mutation of the Asp111 residue. The activation and thermodynamic parameters were also determined from the temperature- and pH-dependent multiwavelength kinetics, and the results are discussed to unravel the role of Asp111 in the mechanism of formation of the dinuclear CuA center in cytochrome c oxidase.

In vitro maturation of NiSOD reveals a role for cytoplasmic histidine in processing and metalation.

The importance of cellular low molecular weight ligands in metalloenzyme maturation is largely unexplored. Maturation of NiSOD requires post-translational N-terminal processing of the proenzyme, SodN, by its cognate protease, SodX. Here we provide evidence for the participation of L-histidine in the protease-dependent maturation of nickel-dependent superoxide dismutase (NiSOD) from Streptomyces coelicolor. In vitro studies using purified proteins cloned from S. coelicolor and overexpressed in E. coli support a model where a ternary complex formed between the substrate (SodN), the protease (SodX) and L-Histidine creates a novel Ni-binding site that is capable of the N-terminal processing of SodN and specifically incorporates Ni into the apo-NiSOD product. Thus, L-Histidine serves many of the functions associated with a metallochaperone or, conversely, eliminates the need for a metallochaperone in NiSOD maturation.

Plastic recognition and electrogenic uniport translocation of 1st-, 2nd-, and 3rd-row transition and post-transition metals by primary-active transmembrane P1B-2-type ATPase pumps.

Transmembrane P1B-type ATPase pumps catalyze the extrusion of transition metal ions across cellular lipid membranes to maintain essential cellular metal homeostasis and detoxify toxic metals. Zn(ii)-pumps of the P1B-2-type subclass, in addition to Zn2+, select diverse metals (Pb2+, Cd2+ and Hg2+) at their transmembrane binding site and feature promiscuous metal-dependent ATP hydrolysis in the presence of these metals. Yet, a comprehensive understanding of the transport of these metals, their relative translocation rates, and transport mechanism remain elusive. We developed a platform for the characterization of primary-active Zn(ii)-pumps in proteoliposomes to study metal selectivity, translocation events and transport mechanism in real-time, employing a "multi-probe" approach with fluorescent sensors responsive to diverse stimuli (metals, pH and membrane potential). Together with atomic-resolution investigation of cargo selection by X-ray absorption spectroscopy (XAS), we demonstrate that Zn(ii)-pumps are electrogenic uniporters that preserve the transport mechanism with 1st-, 2nd- and 3rd-row transition metal substrates. Promiscuous coordination plasticity, guarantees diverse, yet defined, cargo selectivity coupled to their translocation.

The structure of the high-affinity nickel-binding site in the Ni,Zn-HypA•UreE2 complex.

The maturation pathway for the nickel-dependent enzyme urease utilizes the protein UreE as a metallochaperone to supply Ni(II) ions. In Helicobacter pylori urease maturation also requires HypA and HypB, accessory proteins that are commonly associated with hydrogenase maturation. Herein we report on the characterization of a protein complex formed between HypA and the UreE2 dimer. Nuclear magnetic resonance (NMR) coupled with molecular modelling show that the protein complex apo, Zn-HypA•UreE2, forms between the rigorously conserved Met-His-Glu (MHE motif) Ni-binding N-terminal sequence of HypA and the two conserved His102A and His102B located at the dimer interface of UreE2. This complex forms in the absence of Ni(II) and is supported by extensive protein contacts that include the use of the C-terminal sequences of UreE2 to form additional strands of ß-sheet with the Ni-binding domain of HypA. The Ni-binding properties of apo, Zn-HypA•UreE2 and the component proteins were investigated by isothermal titration calorimetry using a global fitting strategy that included all of the relevant equilibria, and show that the Ni,Zn-HypA•UreE2 complex contains a single Ni(II)-binding site with a sub-nanomolar KD. The structural features of this novel Ni(II) site were elucidated using proteins produced with specifically deuterated amino acids, protein point mutations, and the analyses of X-ray absorption spectroscopy, hyperfine shifted NMR features, as well as molecular modeling coupled with quantum-mechanical calculations. The results show that the complex contains a six-coordinate, high-spin Ni(II) site with ligands provided by both component proteins.

Targeted neurorehabilitation strategies in post-stroke aphasia.

BACKGROUND: Aphasia is a debilitating language impairment, affecting millions of people worldwide. About 40% of stroke survivors develop chronic aphasia, resulting in life-long disability. OBJECTIVE: This review examines extrinsic and intrinsic neuromodulation techniques, aimed at enhancing the effects of speech and language therapies in stroke survivors with aphasia. METHODS: We discuss the available evidence supporting the use of transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation, and functional MRI (fMRI) real-time neurofeedback in aphasia rehabilitation. RESULTS: This review systematically evaluates studies focusing on efficacy and implementation of specialized methods for post-treatment outcome optimization and transfer to functional skills. It considers stimulation target determination and various targeting approaches. The translation of neuromodulation interventions to clinical practice is explored, emphasizing generalization and functional communication. The review also covers real-time fMRI neurofeedback, discussing current evidence for efficacy and essential implementation parameters. Finally, we address future directions for neuromodulation research in aphasia. CONCLUSIONS: This comprehensive review aims to serve as a resource for a broad audience of researchers and clinicians interested in incorporating neuromodulation for advancing aphasia care.

Carriage and within-host diversity of mcr-1.1-harbouring Escherichia coli from pregnant mothers: inter- and intra-mother transmission dynamics of mcr-1.1.

Exchange of antimicrobial resistance genes via mobile genetic elements occur in the gut which can be transferred from mother to neonate during birth. This study is the first to analyse transmissible colistin resistance gene, mcr, in pregnant mothers and neonates. Samples were collected from pregnant mothers (rectal) and septicaemic neonates (rectal and blood) and analysed for the presence of mcr, its transmissibility, genome diversity, and exchange of mcr between isolates within an individual and across different individuals (not necessarily mother-baby pairs). mcr-1.1 was detected in rectal samples of pregnant mothers (n = 10, 0.9%), but not in neonates. All mcr-positive mothers gave birth to healthy neonates from whom rectal specimen were not collected. Hence, the transmission of mcr between these mother-neonate pairs could not be studied. mcr-1.1 was noted only in Escherichia coli (phylogroup A & B1), and carried few resistance and virulence genes. Isolates belonged to diverse sequence types (n = 11) with two novel STs (ST12452, ST12455). mcr-1.1 was borne on conjugative IncHI2 bracketed between ISApl1 on Tn6630, and the plasmids exhibited similarities in sequences across the study isolates. Phylogenetic comparison showed that study isolates were related to mcr-positive isolates of animal origin from Southeast Asian countries. Spread of mcr-1.1 within this study occurred either via similar mcr-positive clones or similar mcr-bearing plasmids in mothers. Though this study could not build evidence for mother-baby transmission but the presence of such genes in the maternal specimen may enhance the chances of transmission to neonates.

Pro5 is not essential for the formation of 'Ni-hook' in nickel superoxide dismutase.

The N-terminus of nickel-dependent superoxide dismutase (NiSOD) forms a structural motif known as the "Ni-hook," where the peptide wraps around the metal to bring cysteine-2 and cysteine-6 into spatial proximity, allowing these residues to coordinate in a cis-geometry. A highly conserved proline-5 residue in the Ni-hook adopts a cis-conformation that is widely considered important for its formation. Herein, we investigate this role by point mutation of Pro5 to alanine. The results obtained show that the variant exhibits wild-type-like redox catalysis and features a Ni(III) center very similar to that found in enzyme. Structural analysis using X-ray absorption spectroscopy of the nickel sites in as-isolated P5A-NiSOD reveals changes in the variant and are consistent with a six-coordinate Ni site with (N/O)4S2 coordination. These changes are attributed to changes in the Ni(II) site structure. Nickel-binding studies using isothermal titration calorimetry reveal two binding events with Kd = 25(20) nM, and 250(60) nM. These events are attributed to i) Ni(II) binding to a preformed Ni-hook containing cis-Pro5 and ii) the combination of trans- to cis- isomerization upon Ni(II) binding, respectively. The higher-affinity binding event is absent in P5A-NiSOD, an observation attributed to the low abundance of the cis-Ala5 isomer in the apo-protein.

Capsaicin Inhibits Shigella flexneri Intracellular Growth by Inducing Autophagy.

Antibiotic treatment plays an essential role in preventing Shigella infection. However, incidences of global rise in antibiotic resistance create a major challenge to treat bacterial infection. In this context, there is an urgent need for newer approaches to reduce S. flexneri burden. This study largely focuses on the role of the herbal compound capsaicin (Caps) in inhibiting S. flexneri growth and evaluating the molecular mechanism behind bacterial clearance. Here, we show for the first time that Caps inhibits intracellular S. flexneri growth by inducing autophagy. Activation of autophagy by Caps is mediated through transcription factor TFEB, a master regulator of autophagosome biogenesis. Caps induced the nuclear localization of TFEB. Activation of TFEB further induces the gene transcription of autophagosomal genes. Our findings revealed that the inhibition of autophagy by silencing TFEB and Atg5 induces bacterial growth. Hence, Caps-induced autophagy is one of the key factors responsible for bacterial clearance. Moreover, Caps restricted the intracellular proliferation of S. flexneri-resistant strain. The efficacy of Caps in reducing S. flexneri growth was confirmed by an animal model. This study showed for the first time that S. flexneri infection can be inhibited by inducing autophagy. Overall observations suggest that Caps activates TFEB to induce autophagy and thereby combat S. flexneri infection.