Enzyme Patterns and Factors Associated with Mortality among Patients with Carbapenem Resistant AcinetobacterBaumannii (CRAB) Bacteremia: Real World Evidence from a Tertiary Center in India.

Introduction: In the Indian setting, antimicrobial resistance in A. baumannii is a considerable problem, especially in intensive care units (ICUs). Due to the limited data, clinicians are left with very few choices except polymyxins for treating serious infections caused by A. baumannii. There is sparse data regarding the local mechanisms of resistance. Given the current therapeutic challenges, it is critical to know the local enzymatic patterns and antibiograms. Materials and methods: A retrospective analysis of 50 episodes of bacteremia caused by CRAB. We analyzed the enzyme patterns and the susceptibility rates to various antibiotics. Results: The resistance rates for amikacin, tigecycline, minocycline, and fluoroquinolones were 88, 82, 50, and 88% respectively. OXA-23 was the most commonly isolated enzyme (86% of the isolates produced OXA-23) followed by OXA-51 and NDM. The overall mortality was high (58%). On univariate analysis, pneumonia, and higher Pitt's bacteremia score were significantly associated with mortality (p = 0.04 and p = 0.001 respectively). Of the total patients who received combination therapy, a majority (58%) received polymyxin plus meropenem. Combination therapy using polymyxins as a backbone was not associated with reduced mortality (p = 0.1). Conclusion: A. baumannii is associated with significant morbidity and mortality, as shown in our study. The rates of resistance for aminoglycosides were very high, and minocycline showed better susceptibility rates in comparison with tigecycline. In our study, OXA-23 and NDM remained the most important enzymes. The routine use of the combination of polymyxin and meropenem may not offer a significant advantage over monotherapy. How to cite this article: Prayag PS, Patwardhan SA, Joshi RS, Panchakshari SP, Rane T, Prayag AP. Enzyme Patterns and Factors Associated with Mortality among Patients with Carbapenem Resistant Acinetobacter Baumannii (CRAB) Bacteremia: Real World Evidence from a Tertiary Center in India. Indian J Crit Care Med 2023;27(9):663-668.

Long-COVID cognitive impairments and reproductive hormone deficits in men may stem from GnRH neuronal death.

BACKGROUND: We have recently demonstrated a causal link between loss of gonadotropin-releasing hormone (GnRH), the master molecule regulating reproduction, and cognitive deficits during pathological aging, including Down syndrome and Alzheimer's disease. Olfactory and cognitive alterations, which persist in some COVID-19 patients, and long-term hypotestosteronaemia in SARS-CoV-2-infected men are also reminiscent of the consequences of deficient GnRH, suggesting that GnRH system neuroinvasion could underlie certain post-COVID symptoms and thus lead to accelerated or exacerbated cognitive decline. METHODS: We explored the hormonal profile of COVID-19 patients and targets of SARS-CoV-2 infection in post-mortem patient brains and human fetal tissue. FINDINGS: We found that persistent hypotestosteronaemia in some men could indeed be of hypothalamic origin, favouring post-COVID cognitive or neurological symptoms, and that changes in testosterone levels and body weight over time were inversely correlated. Infection of olfactory sensory neurons and multifunctional hypothalamic glia called tanycytes highlighted at least two viable neuroinvasion routes. Furthermore, GnRH neurons themselves were dying in all patient brains studied, dramatically reducing GnRH expression. Human fetal olfactory and vomeronasal epithelia, from which GnRH neurons arise, and fetal GnRH neurons also appeared susceptible to infection. INTERPRETATION: Putative GnRH neuron and tanycyte dysfunction following SARS-CoV-2 neuroinvasion could be responsible for serious reproductive, metabolic, and mental health consequences in long-COVID and lead to an increased risk of neurodevelopmental and neurodegenerative pathologies over time in all age groups. FUNDING: European Research Council (ERC) grant agreements No 810331, No 725149, No 804236, the European Union Horizon 2020 research and innovation program No 847941, the Fondation pour la Recherche Médicale (FRM) and the Agence Nationale de la Recherche en Santé (ANRS) No ECTZ200878 Long Covid 2021 ANRS0167 SIGNAL, Agence Nationale de la recherche (ANR) grant agreements No ANR-19-CE16-0021-02, No ANR-11-LABEX-0009, No. ANR-10-LABEX-0046, No. ANR-16-IDEX-0004, Inserm Cross-Cutting Scientific Program HuDeCA, the CHU Lille Bonus H, the UK Medical Research Council (MRC) and National Institute of Health and care Research (NIHR).

Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

BACKGROUND: The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. METHODS: nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. RESULTS: In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. CONCLUSIONS: Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

GnRH replacement rescues cognition in Down syndrome.

At the present time, no viable treatment exists for cognitive and olfactory deficits in Down syndrome (DS). We show in a DS model (Ts65Dn mice) that these progressive nonreproductive neurological symptoms closely parallel a postpubertal decrease in hypothalamic as well as extrahypothalamic expression of a master molecule that controls reproduction-gonadotropin-releasing hormone (GnRH)-and appear related to an imbalance in a microRNA-gene network known to regulate GnRH neuron maturation together with altered hippocampal synaptic transmission. Epigenetic, cellular, chemogenetic, and pharmacological interventions that restore physiological GnRH levels abolish olfactory and cognitive defects in Ts65Dn mice, whereas pulsatile GnRH therapy improves cognition and brain connectivity in adult DS patients. GnRH thus plays a crucial role in olfaction and cognition, and pulsatile GnRH therapy holds promise to improve cognitive deficits in DS.

Biological Models of the Lower Human Airways-Challenges and Special Requirements of Human 3D Barrier Models for Biomedical Research.

In our review, we want to summarize the current status of the development of airway models and their application in biomedical research. We start with the very well characterized models composed of cell lines and end with the use of organoids. An important aspect is the function of the mucus as a component of the barrier, especially for infection research. Finally, we will explain the need for a nondestructive characterization of the barrier models using TEER measurements and live cell imaging. Here, organ-on-a-chip technology offers a great opportunity for the culture of complex airway models.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

Leptin brain entry via a tanycytic LepR-EGFR shuttle controls lipid metabolism and pancreas function.

Metabolic health depends on the brain's ability to control food intake and nutrient use versus storage, processes that require peripheral signals such as the adipocyte-derived hormone, leptin, to cross brain barriers and mobilize regulatory circuits. We have previously shown that hypothalamic tanycytes shuttle leptin into the brain to reach target neurons. Here, using multiple complementary models, we show that tanycytes express functional leptin receptor (LepR), respond to leptin by triggering Ca2+ waves and target protein phosphorylation, and that their transcytotic transport of leptin requires the activation of a LepR-EGFR complex by leptin and EGF sequentially. Selective deletion of LepR in tanycytes blocks leptin entry into the brain, inducing not only increased food intake and lipogenesis but also glucose intolerance through attenuated insulin secretion by pancreatic ß-cells, possibly via altered sympathetic nervous tone. Tanycytic LepRb-EGFR-mediated transport of leptin could thus be crucial to the pathophysiology of diabetes in addition to obesity, with therapeutic implications.

Targeting Trimeric and Tetrameric Proanthocyanidins of Cinnamomum verum Bark as Bioactives for Dental Therapies.

The present study elucidated the structures of three A-type tri- and tetrameric proanthocyanidins (PACs) isolated from Cinnamomum verum bark to the level of absolute configuration and determined their dental bioactivity using two therapeutically relevant bioassays. After selecting a PAC oligomer fraction via a biologically diverse bioassay-guided process, in tandem with centrifugal partition chromatography, phytochemical studies led to the isolation of PAC oligomers that represent the main bioactive principles of C. verum: two A-type tetrameric PACs, epicatechin-(2ß→O→7,4ß→8)-epicatechin-(4ß→6)-epicatechin-(2ß→O→7,4ß→8)-catechin (1) and parameritannin A1 (2), together with a trimer, cinnamtannin B1 (3). Structure determination of the underivatized proanthocyanidins utilized a combination of HRESIMS, ECD, 1D/2D NMR, and 1H iterative full spin analysis data and led to NMR-based evidence for the deduction of absolute configuration in constituent catechin and epicatechin monomeric units.

A dynamic mechanical method to assess bulk viscoelastic behavior of the dentin extracellular matrix.

OBJECTIVES: To develop a protocol for assessment of the bulk viscoelastic behavior of dentin extracellular matrix (ECM), and to assess relationships between induced collagen cross-linking and viscoelasticity of the dentin ECM. METHODS: Dentin ECM was treated with agents to induce exogenous collagen cross-linking: proanthocyanidins (PACs) from Vitis vinifera - VVe, PACs from Pinus massoniana - PMe, glutaraldehyde - (GA), or kept untreated (control). A dynamic mechanical strain sweep method was carried out in a 3-point bending submersion clamp at treatment; after protein destabilization with 4 M urea and after 7-day, 6-month, and 12-month incubation in simulated body fluid. Tan δ, storage (E'), loss (E"), and complex moduli (E*) were calculated and data were statistically analyzed using two-way ANOVA and post-hoc tests (α = 0.05). Chemical analysis of dentin ECM before and after protein destabilization was assessed with ATR-FTIR spectroscopy. RESULTS: Significant interactions between study factors (treatment vs. time points, p < 0.001) were found for all viscoelastic parameters. Despite a significant decrease in all moduli after destabilization, PAC-treated dentin remained statistically higher than control (p < 0.001), indicating permanent mechanical enhancement after biomodification. Covalently crosslinked, GA-treated dentin was unaffected by destabilization (p = 0.873) and showed the lowest damping capacity (tan δ) at all time points (p < 0.001). After 12 months, the damping capacity of PMe and VVe groups decreased significantly. Changes in all amide IR resonances revealed a partial chemical reversal of PAC-mediated biomodification. SIGNIFICANCE: Viscoelastic measurements and IR spectroscopy aid in elucidating the role of inter-molecular collagen cross-linking in the mechanical behavior of dentin ECM.

Neuropilin-1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction.

Hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH), the "master molecule" regulating reproduction and fertility, migrate from their birthplace in the nose to their destination using a system of guidance cues, which include the semaphorins and their receptors, the neuropilins and plexins, among others. Here, we show that selectively deleting neuropilin-1 in new GnRH neurons enhances their survival and migration, resulting in excess neurons in the hypothalamus and in their unusual accumulation in the accessory olfactory bulb, as well as an acceleration of mature patterns of activity. In female mice, these alterations result in early prepubertal weight gain, premature attraction to male odors, and precocious puberty. Our findings suggest that rather than being influenced by peripheral energy state, GnRH neurons themselves, through neuropilin-semaphorin signaling, might engineer the timing of puberty by regulating peripheral adiposity and behavioral switches, thus acting as a bridge between the reproductive and metabolic axes.

Tri- and Tetrameric Proanthocyanidins with Dentin Bioactivities from Pinus massoniana.

Guided by dentin biomechanical bioactivity, this phytochemical study led to the elucidation of an extended set of structurally demanding proanthocyanidins (PACs). Unambiguous structure determination involved detailed spectroscopic and chemical characterization of four A-type dimers (2 and 4-6), seven trimers (10-16), and six tetramers (17-22). New outcomes confirm the feasibility of determining the absolute configuration of the catechol monomers in oligomeric PACs by one-dimensional (1D) and two-dimensional (2D) NMR. Electronic circular dichroism as well as phloroglucinolysis followed by mass spectrometry and chiral phase high-performance liquid chromatography (HPLC) analysis generated the necessary chiral reference data. In the context of previously reported dentin-bioactive PACs, accurately and precisely assigned 13C NMR resonances enabled absolute stereochemical assignments of PAC monomers via (i) inclusion of the 13C NMR γ-gauche effect and (ii) determination of differential 13C chemical shift values (ΔδC) in comparison with those of the terminal monomer (unit II) in the dimers 2 and 4-6. Among the 13 fully elucidated PACs, eight were identified as new, and one structure (11) was revised based on new knowledge gained regarding the subtle, stereospecific spectroscopic properties of PACs.

Differential activation of the mTOR/autophagy pathway predicts cognitive performance in APP/PS1 mice.

The molecular bases underlying cognitive impairments in Alzheimer's disease remain elusive. In this study, we sought to determine the molecular correlates of memory deficits in APP/PS1 mice, a widely used animal model of Alzheimer's disease. To this end, we tested 18-month-old APP/PS1 mice in the Morris water maze and ranked them by their spatial memory performance. We found that some APP/PS1 mice performed poorly, whereas others performed as well as nontransgenic mice. We took advantage of this intragroup variability to identify the best predictor of cognitive deficits. In this APP/PS1 cohort, soluble and insoluble amyloid-ß levels did not correlate significantly with cognitive performance. However, we found that cognitive performance within the APP/PS1 group had a strong inverse correlation with Aß plaque load and mammalian target of rapamycin activation and positively correlated with autophagy activation. Our data suggest that mammalian target of rapamycin signaling may account cognitive performance in APP/PS1 mice.

Proanthocyanidin Dimers and Trimers from Vitis vinifera Provide Diverse Structural Motifs for the Evaluation of Dentin Biomodification.

Aimed at exploring the dentin biomodification potential of proanthocyanidins (PACs) for the development of dental biomaterials, this study reports the phytochemical and dental evaluation of nine B-type PACs from grape seed extract (GSE). Out of seven isolated dimers (1-7), four new compounds (2, 3, 5, and 6) involved relatively rare ent-catechin or ent-epicatechin monomeric flavan-3-ol units. Low-temperature NMR analyses conducted along with phloroglucinolysis and electronic circular dichroism enabled unequivocal structural characterization and stereochemical assignment. Additionally, one known (8) and one new (9) B-type trimer were characterized. Differential 13C NMR chemical shifts (Δδ) were used to determine the absolute configuration of 9, relative to the dimers 1 and 2 as the possible constituent subunits. Compared to the dimers, the trimers showed superior dentin biomodification properties. The dimers, 1-7, exhibited pronounced differences in their collagenase inhibitory activity, while enhancing dentin stiffness comparably. This suggests that PAC structural features such as the degree of polymerization, relative and absolute configuration have a differential influence on enhancement of dentin biomechanical and biostability. As mechanical enhancement to dentin and resistance to proteolytic biodegradation are both essential properties functional and stable dentin substrate, the structurally closely related PACs suggest a new metric, the dentin biomodification potential (DBMP) that may rationalize both properties.

Evidence to the role of interflavan linkages and galloylation of proanthocyanidins at sustaining long-term dentin biomodification.

OBJECTIVES: The interactivity of proanthocyanidins (PACs) with collagen modulates dentin matrix biomechanics and biostability. Herein, PAC extracts selected based on structural diversity were investigated to determine key PAC features driving sustained effects on dentin matrices over a period of 18months. METHODS: The chemical profiles of PAC-rich plant sources, Pinus massoniana (PM), Cinnamomum verum (CV) and Hamamelis virginiana (HV) barks, as well as Vitis vinifera (VV) seeds, were obtained by diol HPLC analysis after partitioning of the extracts between methyl acetate and water. Dentin matrices (n=15) were prepared from human molars to determine the apparent modulus of elasticity over 18months of aging. Susceptibility of the dentin matrix to degradation by endogenous and exogenous proteases was determined by presence of solubilized collagen in supernatant, and resistance to degradation by bacterial collagenase, respectively. Data were analyzed using ANOVA and Games-Howell post hoc tests (α=0.05). RESULTS: After 18months, dentin matrices modified by PM and CV extracts, containing only non-galloylated PACs, were highly stable mechanically (p<0.05). Dentin matrices treated with CV exhibited the lowest degradation by bacterial collagenase after 1h and 18months of aging (p<0.05), while dentin matrices treated with PM showed the least mass loss and collagen solubilization by endogenous enzymes over time (p<0.05). SIGNIFICANCE: Resistance against long-term degradation was observed for all experimental groups; however, the most potent and long-lasting dentin biomodification resulted from non-galloylated PACs.

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development.

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

Absolute Configuration of Native Oligomeric Proanthocyanidins with Dentin Biomodification Potency.

The structurally complex oligomeric proanthocyanidins (OPACs) are promising biomimetic agents, capable of strengthening the macromolecular backbone of teeth via intermolecular and intermicrofibrillar cross-linking. This study establishes analytical methods capable of determining the absolute configuration of the catechin-type monomeric units of underivatized OPACs. This preserves the capacity of their biological evaluation, aimed at understanding the inevitably stereospecific interactions between the OPACs and dentin collagen. Guided by dental bioassays (modulus of elasticity, long-term stability), two new trimeric and tetrameric A-type OPACs were discovered as dentin biomodifiers from pine (Pinus massoniana) bark: epicatechin-(2ß→O→7,4ß→8)-epicatechin-(2ß→O→7,4ß→8)-catechin (5) and epicatechin-(2ß→O→7,4ß→8)-epicatechin-(2ß→O→7,4ß→6)-epicatechin-(2ß→O→7,4ß→8)-catechin (6), respectively. Combining 1D/2D NMR, HRESIMS, ECD, 1H iterative full spin analysis (HiFSA), and gauge-invariant atomic orbital (GIAO) δ calculations, we demonstrate how 13C NMR chemical shifts (diastereomeric building blocks (A-type dimers)) empower the determination of the absolute configuration of monomeric units in the higher oligomers 5 and 6. Collectively, NMR with ECD reference data elevates the level of structural information achievable for these structurally demanding molecules when degradation analysis is to be avoided. Considering their numerous and deceptively subtle, but 3D impactful, structural variations, this advances the probing of OPAC chemical spaces for species that bind selectively to collagenous and potentially other biologically important biomacromolecules.

Evolution of Quantitative Measures in NMR: Quantum Mechanical qHNMR Advances Chemical Standardization of a Red Clover (Trifolium pratense) Extract.

Chemical standardization, along with morphological and DNA analysis ensures the authenticity and advances the integrity evaluation of botanical preparations. Achievement of a more comprehensive, metabolomic standardization requires simultaneous quantitation of multiple marker compounds. Employing quantitative 1H NMR (qHNMR), this study determined the total isoflavone content (TIfCo; 34.5-36.5% w/w) via multimarker standardization and assessed the stability of a 10-year-old isoflavone-enriched red clover extract (RCE). Eleven markers (nine isoflavones, two flavonols) were targeted simultaneously, and outcomes were compared with LC-based standardization. Two advanced quantitative measures in qHNMR were applied to derive quantities from complex and/or overlapping resonances: a quantum mechanical (QM) method (QM-qHNMR) that employs 1H iterative full spin analysis, and a non-QM method that uses linear peak fitting algorithms (PF-qHNMR). A 10 min UHPLC-UV method provided auxiliary orthogonal quantitation. This is the first systematic evaluation of QM and non-QM deconvolution as qHNMR quantitation measures. It demonstrates that QM-qHNMR can account successfully for the complexity of 1H NMR spectra of individual analytes and how QM-qHNMR can be built for mixtures such as botanical extracts. The contents of the main bioactive markers were in good agreement with earlier HPLC-UV results, demonstrating the chemical stability of the RCE. QM-qHNMR advances chemical standardization by its inherent QM accuracy and the use of universal calibrants, avoiding the impractical need for identical reference materials.