Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells.

In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD.

ArmA and RmtB Were the Predominant 16S RMTase Genes Responsible for Aminoglycoside-resistant Isolates in Korea.

Pathogenic gram-negatives that produce 16S ribosomal RNA methyltransferases (16S RMTases) have already been distributed all over the world. To investigate the predominance of aminoglycoside resistance associated with 16S RMTases in Korea, we collected a total of 222 amikacin resistant Gram-negative clinical isolates from patient specimens between 1999 and 2015 from three hospital banks across Korea. ArmA and rmtB were the predominant 16S RMTase genes responsible for aminoglycoside-resistant isolates circulating in Korean community settings although only one rmtA-producing isolate was detected in 2006.

Structural and mechanistic insights into the inhibition of class C ß-lactamases through the adenylylation of the nucleophilic serine.

Objectives: : Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C ß-lactamases. Methods: : The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5'-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates. Results: : The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies ( k inact / K I ) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M -1 s -1 , respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria. Conclusions: : Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C ß-lactamase inhibitors on the basis of the nucleotide scaffold.

Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca(2+) concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca(2+) levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death.

Cross-protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv.

Gram-negative Vibrio species secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross-reactivity and cross-protectivity of mAbs against V. vulnificus RtxA1/MARTXVv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTXVc , inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins.

Development and characterization of a catalytically inactive cysteine protease domain of RtxA1/MARTXVv as a potential vaccine for Vibrio vulnificus.

Recent studies have defined several virulence factors as vaccine candidates against Vibrio vulnificus. However, most of these factors have the potential to cause pathogenic effects in the vaccinees or induce incomplete protection. To overcome these drawbacks, a catalytically inactive form, CPDVv (C3725S), of the well-conserved cysteine protease domain (CPD) of V. vulnificus multifunctional autoprocessing repeats-in-toxin (MARTXVv /RtxA1) was recombinantly generated and characterized. Notably, active and passive immunization with CPDVv (C3725S) conferred protective immunity against V. vulnificus strains. These results may provide a novel framework for developing safe and efficient subunit vaccines and/or therapeutics against V. vulnificus that target the CPD of MARTX toxins.

Acute Cholecystitis in Patients with Scrub Typhus.

Acute cholecystitis is a rare complication of scrub typhus. Although a few such cases have been reported in patients with scrub typhus, the clinical course is not well described. Of 12 patients, acute cholecystitis developed in 66.7% (8/12) of patients older than 60 yr. The scrub typhus group with acute cholecystitis had marginal significant longer hospital stay and higher cost than the group without cholecystitis according to propensity score matching. Scrub typhus should be kept in mind as a rare etiology of acute cholecystitis in endemic areas because the typical signs of scrub typhus such as skin rash and eschar can present after the abdominal pain.

Comparison of Arbekacin and Vancomycin in Treatment of Chronic Suppurative Otitis Media by Methicillin Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of ear infections. We attempted to evaluate the clinical usefulness of arbekacin in treating chronic suppurative otitis media (CSOM) by comparing its clinical efficacy and toxicity with those of vancomycin. Efficacy was classified according to bacterial elimination or bacteriologic failure and improved or failed clinical efficacy response. Ninety-five subjects were diagnosed with CSOM caused by MRSA. Twenty of these subjects were treated with arbekacin, and 36 with vancomycin. The bacteriological efficacy (bacterial elimination, arbekacin vs. vancomycin: 85.0% vs. 97.2%) and improved clinical efficacy (arbekacin vs. vancomycin; 90.0% vs. 97.2%) were not different between the two groups. However, the rate of complications was higher in the vancomycin group (33.3%) than in the arbekacin group (5.0%) (P=0.020). In addition, a total of 12 adverse reactions were observed in the vancomycin group; two for hepatotoxicity, one for nephrotoxicity, eight for leukopenia, two for skin rash, and one for drug fever. It is suggested that arbekacin be a good alternative drug to vancomycin in treatment of CSOM caused by MRSA.

Monoclonal antibodies against Vibrio vulnificus RtxA1 elicit protective immunity through distinct mechanisms.

Vibrio vulnificus causes rapidly progressing septicemia with an extremely high mortality rate (≥50%), even with aggressive antibiotic treatment. The bacteria secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins, which are involved in the pathogenesis of Gram-negative Vibrio species. Recently, we reported that immunization with the C-terminal region of V. vulnificus RtxA1/MARTXVv, RtxA1-C, elicits a protective immune response against V. vulnificus through a poorly defined mechanism. In this study, we generated a panel of new monoclonal antibodies (MAbs) against V. vulnificus RtxA1-C and investigated their protective efficacies and mechanisms in a mouse model of infection. Prophylactic administration of seven MAbs strongly protected mice against lethal V. vulnificus infection (more than 90% survival). Moreover, three of these MAbs (21RA, 24RA, and 47RA) demonstrated marked efficacy as postexposure therapy. Notably, 21RA was therapeutically effective against lethal V. vulnificus infection by a variety of routes. Using Fab fragments and a neutropenic mouse model, we showed that 21RA and 24RA mediate protection from V. vulnificus infection through an Fc-independent and/or neutrophil-independent pathway. In contrast, 47RA-mediated protection was dependent on its Fc region and was reduced to 50% in neutropenic mice compared with 21RA-mediated and 24RA-mediated protection. Bacteriological study indicated that 21RA appears to enhance the clearance of V. vulnificus from the blood. Overall, these studies suggest that humoral immunity controls V. vulnificus infection through at least two different mechanisms. Furthermore, our panel of MAbs could provide attractive candidates for the further development of immunoprophylaxis/therapeutics and other therapies against V. vulnificus that target the MARTX toxin.

ATP-binding mode including a carbamoylated lysine and two Mg(2+) ions, and substrate-binding mode in Acinetobacter baumannii MurF.

MurF adds d-Ala-d-Ala dipeptide to UDP-N-acetylmuramyl-l-Ala-γ-d-Glu-m-DAP (or l-Lys) in an ATP-dependent manner, which is the last step in the biosynthesis of monomeric precursor of peptidoglycan. Here we report crystal structures of two MurF-ATP complexes: the MurF-ATP complex and the MurF-ATP-UDP complex. The ATP-binding mode revealed by the crystal structure of the MurF-ATP complex confirms the previous biochemical demonstration that a carbamoylated lysine and two Mg(2+) ions are required for enzyme activity of MurF. The UDP-MurF interactions observed in the crystal structure of the MurF-ATP-UDP complex depict the characteristic substrate-binding mode of MurF. The emergence and dissemination of multidrug-resistant Acinetobacter baumannii strains are great threats to public health. Therefore, the structural information on A. baumannii MurF as a validated target for drug discovery will provide a framework to develop antibacterial agents against multidrug-resistant A. baumannii infections as well as to understand the reaction mechanism of MurF.

Predictive Modeling and Integrated Risk Assessment of Postoperative Mortality and Pneumonia in Traumatic Brain Injury Patients through Clustering and Machine Learning: Retrospective Study.

This study harnessed machine learning to forecast postoperative mortality (POM) and postoperative pneumonia (PPN) among surgical traumatic brain injury (TBI) patients. Our analysis centered on the following key variables: Glasgow Coma Scale (GCS), midline brain shift (MSB), and time from injury to emergency room arrival (TIE). Additionally, we introduced innovative clustered variables to enhance predictive accuracy and risk assessment. Exploring data from 617 patients spanning 2012 to 2022, we observed that 22.9% encountered postoperative mortality, while 30.0% faced postoperative pneumonia (PPN). Sensitivity for POM and PPN prediction, before incorporating clustering, was in the ranges of 0.43-0.82 (POM) and 0.54-0.76 (PPN). Following clustering, sensitivity values were 0.47-0.76 (POM) and 0.61-0.77 (PPN). Accuracy was in the ranges of 0.67-0.76 (POM) and 0.70-0.81 (PPN) prior to clustering and 0.42-0.73 (POM) and 0.55-0.73 (PPN) after clustering. Clusters characterized by low GCS, small MSB, and short TIE exhibited a 3.2-fold higher POM risk compared to clusters with high GCS, small MSB, and short TIE. In summary, leveraging clustered variables offers a novel avenue for predicting POM and PPN in TBI patients. Assessing the amalgamated impact of GCS, MSB, and TIE characteristics provides valuable insights for clinical decision making.

CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

Gene expression is changed by disease, but how these molecular responses arise and contribute to pathophysiology remains less understood. We discover that ß-amyloid, a trigger of Alzheimer's disease (AD), promotes the formation of pathological CREB3L2-ATF4 transcription factor heterodimers in neurons. Through a multilevel approach based on AD datasets and a novel chemogenetic method that resolves the genomic binding profile of dimeric transcription factors (ChIPmera), we find that CREB3L2-ATF4 activates a transcription network that interacts with roughly half of the genes differentially expressed in AD, including subsets associated with ß-amyloid and tau neuropathologies. CREB3L2-ATF4 activation drives tau hyperphosphorylation and secretion in neurons, in addition to misregulating the retromer, an endosomal complex linked to AD pathogenesis. We further provide evidence for increased heterodimer signaling in AD brain and identify dovitinib as a candidate molecule for normalizing ß-amyloid-mediated transcriptional responses. The findings overall reveal differential transcription factor dimerization as a mechanism linking disease stimuli to the development of pathogenic cellular states.

Deep Learning-Based Knee MRI Classification for Common Peroneal Nerve Palsy with Foot Drop.

Foot drop can have a variety of causes, including the common peroneal nerve (CPN) injuries, and is often difficult to diagnose. We aimed to develop a deep learning-based algorithm that can classify foot drop with CPN injury in patients with knee MRI axial images only. In this retrospective study, we included 945 MR image data from foot drop patients confirmed with CPN injury in electrophysiologic tests (n = 42), and 1341 MR image data with non-traumatic knee pain (n = 107). Data were split into training, validation, and test datasets using a 8:1:1 ratio. We used a convolution neural network-based algorithm (EfficientNet-B5, ResNet152, VGG19) for the classification between the CPN injury group and the others. Performance of each classification algorithm used the area under the receiver operating characteristic curve (AUC). In classifying CPN MR images and non-CPN MR images, EfficientNet-B5 had the highest performance (AUC = 0.946), followed by the ResNet152 and the VGG19 algorithms. On comparison of other performance metrics including precision, recall, accuracy, and F1 score, EfficientNet-B5 had the best performance of the three algorithms. In a saliency map, the EfficientNet-B5 algorithm focused on the nerve area to detect CPN injury. In conclusion, deep learning-based analysis of knee MR images can successfully differentiate CPN injury from other etiologies in patients with foot drop.

A cross-protective mAb recognizes a novel epitope within the flavivirus NS1 protein.

Despite a resurgence of flavivirus infections worldwide, no approved therapeutic agent exists for any member of the genus. While cross-reactive antibodies with therapeutic potential against flaviviruses have been generated, the majority of them are anti-E antibodies with the potential to cause antibody-dependent enhancement of flavivirus infection and disease. We described previously mAbs against the non-structural NS1 protein of the West Nile virus (WNV) that were protective in mice when administered pre- or post-infection of WNV. Here, we demonstrate that one of these mAbs (16NS1) cross-reacted with Japanese encephalitis virus (JEV) and exhibited protective activity against a lethal JEV infection. Overlapping peptide mapping analysis combined with site-specific mutations identified a novel epitope ¹¹6KAWGKSILFA¹²5 and critical amino acid residues (¹¹8W and ¹²²I) for 16NS1 mAb binding. These results may facilitate the development of a broadly therapeutic mAb that lacks enhancing potential and/or subunit-based vaccine against flaviviruses that target the NS1 protein.

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus.

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.

A systemic cell stress signal confers neuronal resilience toward oxidative stress in a Hedgehog-dependent manner.

Cells possess several conserved adaptive mechanisms to respond to stress. Stress signaling is initiated to reestablish cellular homeostasis, but its effects on the tissue or systemic levels are far less understood. We report that the secreted luminal domain of the endoplasmic reticulum (ER) stress transducer CREB3L2 (which we name TAILS [transmissible activator of increased cell livability under stress]) is an endogenous, cell non-autonomous activator of neuronal resilience. In response to oxidative insults, neurons secrete TAILS, which potentiates hedgehog signaling through direct interaction with Sonic hedgehog (SHH) and its receptor PTCH1, leading to improved antioxidant signaling and mitochondrial function in neighboring neurons. In an in vivo model of ischemic brain injury, administration of TAILS enables survival of CNS neurons and fully preserves cognitive function in behavioral tests. Our findings reveal an SHH-mediated, cell non-autonomous branch of cellular stress signaling that confers resilience to oxidative stress in the mature brain, providing protection from ischemic neurodegeneration.

New Genotypes and Diversity of Orientia tsutsugamushi DNA samples from Patients with Scrub Typhus in South Korea as Determined by Multilocus Sequence Typing.

Orientia tsutsugamushi, an obligate intracellular organism, is the causative agent of scrub typhus. Multilocus sequence typing (MLST) is a genetic typing method that provides a unified bacterial isolate characterization approach. However, there are no comparative studies in South Korea on the genotypic properties of O. tsutsugamushi based on MLST. To conduct a comparative analysis with previous data collected from Thailand, Laos, and Japan, we investigated the genetic diversity of O. tsutsugamushi from 51 patients with scrub typhus in South Korea by using MLST. The MLST analysis revealed 10 new alleles in the housekeeping genes: gpsA, n = 2; mdh, n = 1; nrdB, n = 1; nuoF, n = 1; ppdK, n = 1; sucB, n = 2; and sucD, n = 2. These novel alleles led to the assignment of six new sequence types (STs) (ST93-98). The 51 samples corresponded to seven different STs (ST48 and ST93-98), with ST48 accounting for the largest proportion (49.0%) of O. tsutsugamushi STs in South Korea. Interestingly, O. tsutsugamushi from patients with scrub typhus in South Korea were clustered in two different clades, and the five Korean STs (ST48, ST93, ST94, ST95, and ST98) were close genetically to ST80, which was isolated from Laos. The remaining two STs (ST96 and ST97) were close genetically to ST49 (Ikeda, Japan). Overall, our results suggest that the relative genetic stability and the clonal populations of O. tsutsugamushi strains in South Korea have remained mostly conserved.

Presenilin 2 N141I mutation induces hyperactive immune response through the epigenetic repression of REV-ERBα.

Hyperimmunity drives the development of Alzheimer disease (AD). The immune system is under the circadian control, and circadian abnormalities aggravate AD progress. Here, we investigate how an AD-linked mutation deregulates expression of circadian genes and induces cognitive decline using the knock-in (KI) mice heterozygous for presenilin 2 N141I mutation. This mutation causes selective overproduction of clock gene-controlled cytokines through the DNA hypermethylation-mediated repression of REV-ERBα in innate immune cells. The KI/+ mice are vulnerable to otherwise innocuous, mild immune challenges. The antipsychotic chlorpromazine restores the REV-ERBα level by normalizing DNA methylation through the inhibition of PI3K/AKT1 pathway, and prevents the overexcitation of innate immune cells and cognitive decline in KI/+ mice. These results highlight a pathogenic link between this AD mutation and immune cell overactivation through the epigenetic suppression of REV-ERBα.

Structural basis for the specialization of Nur, a nickel-specific Fur homolog, in metal sensing and DNA recognition.

Nur, a member of the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and anti-oxidative response in Streptomyces coelicolor. Here we report the 2.4-A resolution crystal structure of Nur. It contains a unique nickel-specific metal site in addition to a nonspecific common metal site. The identification of the 6-5-6 motif of the Nur recognition box and a Nur/DNA complex model reveals that Nur mainly interacts with terminal bases of the palindrome on complex formation. This contrasts with more distributed contacts between Fur and the n-1-n type of the Fur-binding motif. The disparity between Nur and Fur in the conformation of the S1-S2 sheet in the DNA-binding domain can explain their different DNA-recognition patterns. Furthermore, the fact that the specificity of Nur in metal sensing and DNA recognition is conferred by the specific metal site suggests that its introduction drives the evolution of Nur orthologs in the Fur family.

GSK3B induces autophagy by phosphorylating ULK1.

Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis.