NPP-669, a Novel Broad-Spectrum Antiviral Therapeutic with Excellent Cellular Uptake, Antiviral Potency, Oral Bioavailability, Preclinical Efficacy, and a Promising Safety Margin.

DNA viruses are responsible for many diseases in humans. Current treatments are often limited by toxicity, as in the case of cidofovir (CDV, Vistide), a compound used against cytomegalovirus (CMV) and adenovirus (AdV) infections. CDV is a polar molecule with poor bioavailability, and its overall clinical utility is limited by the high occurrence of acute nephrotoxicity. To circumvent these disadvantages, we designed nine CDV prodrug analogues. The prodrugs modulate the polarity of CDV with a long sulfonyl alkyl chain attached to one of the phosphono oxygens. We added capping groups to the end of the alkyl chain to minimize ß-oxidation and focus the metabolism on the phosphoester hydrolysis, thereby tuning the rate of this reaction by altering the alkyl chain length. With these modifications, the prodrugs have excellent aqueous solubility, optimized metabolic stability, increased cellular permeability, and rapid intracellular conversion to the pharmacologically active diphosphate form (CDV-PP). The prodrugs exhibited significantly enhanced antiviral potency against a wide range of DNA viruses in infected human foreskin fibroblasts. Single-dose intravenous and oral pharmacokinetic experiments showed that the compounds maintained plasma and target tissue levels of CDV well above the EC50 for 24 h. These experiments identified a novel lead candidate, NPP-669. NPP-669 demonstrated efficacy against CMV infections in mice and AdV infections in hamsters following oral (p.o.) dosing at a dose of 1 mg/kg BID and 0.1 mg/kg QD, respectively. We further showed that NPP-669 at 30 mg/kg QD did not exhibit histological signs of toxicity in mice or hamsters. These data suggest that NPP-669 is a promising lead candidate for a broad-spectrum antiviral compound.

Susceptibility of LoRa Communications to Intentional Electromagnetic Interference with Different Sweep Periods.

This work presents a detailed analysis of the susceptibility of LoRa communications in the presence of intentional jamming signals. The analysis is performed with a periodic frequency-sweeping intentional electromagnetic interference, corresponding to the most common jamming signals. Such a waveform faithfully represents the signals emitted by commercial jammers. As the sweep period of the jamming signals may vary from one such device to another, the analyses are conducted with different sweep period values, from 1 µs to 50 µs. The experimental results indicate that the impact varies significantly according to the sweep period of the jamming signal. The detailed analysis allows us to identify the jamming signals to which LoRa communications can be resilient or not as well as to identify which LoRa channels are less affected during an attack.

On the Stationarity Time of a Vehicle-to-Infrastructure Massive Radio Channel in a Line-of-Sight Suburban Environment.

Massive multiple-input multiple-output (mMIMO) communication systems are a pillar technology for 5G. However, the wireless radio channel models relying on the assumption of wide-sense stationary uncorrelated scattering (WSSUS) may not always be valid for dynamic scenarios. Nonetheless, an analysis of the stationarity time that validates this hypothesis for mMIMO vehicular channels as well as a clear relationship with the scattering properties is missing in the literature. Here, time-varying single-user mMIMO radio channels were measured in a suburban environment at the 5.89 GHz vehicular band with a strong Line-of-Sight (LOS) to study the non-WSSUS and large scale characteristics of the vehicle-to-infrastructure (V2I) link. The generalized local scattering function (GLSF), computed from the sampled channels, was used to derive (1) the spatial distribution of the stationarity time using the channel correlation function (CCF) and empirical collinearity methods and (2) the root mean square delay/angular spread and coherence time/bandwidth values from the projected power delay profile (PDP) and Doppler power spectra (DPS). The results highlight the high degree of correlation between the spatial distribution of the stationarity time and the scattering properties along the measurement route.

Epigenetic Modification 6-Methyladenosine Can Impact the Potency and Specificity of siRNA.

The introduction of N6-methyladenosine (m6 A) into siRNA targeting Factor VII impacts its potency in cells and has a significant influence on the selectivity of siRNA, including reduced off-targeting. These effects are dependent on the position of m6 A in the siRNA duplex, with some of the sequences identified as more potent and/or selective than their non-methylated counterpart. These findings broaden the repertoire of available chemical modifications for siRNA therapeutics and imply potential regulatory role of N6-methyladenosine in the RNAi pathways.

The use of hydrolytic enzymes and multi-stage tandem mass spectrometry to analyze pyridoxal phosphate-modified peptides.

A previous approach was established that allowed direct identification of pyridoxal-5'-phosphate (PLP) bonding sites in proteins using mass spectrometry after tryptic proteolysis. The approach required peptide mass fingerprinting owing to suppressed amide backbone fragmentation in favor of side-chain elimination of diagnostic product ions from PLP-derivatized lysyl residues. While sufficient for purified proteins, unambiguous sequence determination is needed to assign PLP bonding sites in unknown proteins in complex mixtures. Here, we describe the use of hydrolytic enzymes and multi-stage tandem mass spectrometry to elucidate the amino acid sequence and PLP bonding site in PLP-modified peptides.

Transcriptomic characterization of culture-associated changes in murine and human precision-cut tissue slices.

Our knowledge of complex pathological mechanisms underlying organ fibrosis is predominantly derived from animal studies. However, relevance of animal models for human disease is limited; therefore, an ex vivo model of human precision-cut tissue slices (PCTS) might become an indispensable tool in fibrosis research and drug development by bridging the animal-human translational gap. This study, presented as two parts, provides comprehensive characterization of the dynamic transcriptional changes in PCTS during culture by RNA sequencing. Part I investigates the differences in culture-induced responses in murine and human PCTS derived from healthy liver, kidney and gut. Part II delineates the molecular processes in cultured human PCTS generated from diseased liver, kidney and ileum. We demonstrated that culture was associated with extensive transcriptional changes and impacted PCTS in a universal way across the organs and two species by triggering an inflammatory response and fibrosis-related extracellular matrix (ECM) remodelling. All PCTS shared mRNA upregulation of IL-11 and ECM-degrading enzymes MMP3 and MMP10. Slice preparation and culturing activated numerous pathways across all PCTS, especially those involved in inflammation (IL-6, IL-8 and HMGB1 signalling) and tissue remodelling (osteoarthritis pathway and integrin signalling). Despite the converging effects of culture, PCTS display species-, organ- and pathology-specific differences in the regulation of genes and canonical pathways. The underlying pathology in human diseased PCTS endures and influences biological processes like cytokine release. Our study reinforces the use of PCTS as an ex vivo fibrosis model and supports future studies towards its validation as a preclinical tool for drug development.

Novel Formulation Strategy to Improve the Feasibility of Amifostine Administration.

PURPOSE: Amifostine (AMF), a radioprotectant, is FDA-approved for intravenous administration in cancer patients receiving radiation therapy (XRT). Unfortunately, it remains clinically underutilized due to adverse side effects. The purpose of this study is to define the pharmacokinetic profile of an oral AMF formulation potentially capable of reducing side effects and increasing clinical feasibility. METHODS: Calvarial osteoblasts were radiated under three conditions: no drug, AMF, and WR-1065 (active metabolite). Osteogenic potential of cells was measured using alkaline phosphatase staining. Next, rats were given AMF intravenously or directly into the jejunum, and pharmacokinetic profiles were evaluated. Finally, rats were given AMF orally or subcutaneously, and blood samples were analyzed for pharmacokinetics. RESULTS: WR-1065 preserved osteogenic potential of calvarial osteoblasts after XRT to a greater degree than AMF. Direct jejunal AMF administration incurred a systemic bioavailability of 61.5%. Subcutaneously administrated AMF yielded higher systemic levels, a more rapid peak exposure (0.438 vs. 0.875 h), and greater total systemic exposure of WR-1065 (116,756 vs. 16,874 ng*hr/ml) compared to orally administered AMF. CONCLUSIONS: Orally administered AMF achieves a similar systemic bioavailability and decreased peak plasma level of WR-1065 compared to intravenously administered AMF, suggesting oral AMF formulations maintain radioprotective efficacy without causing onerous side effects, and are clinically feasible.

The association of angiogenic factors and chronic kidney disease.

BACKGROUND: There are limited data on the associations of circulating angiogenic factors with chronic kidney disease (CKD). We investigate the associations of circulating vascular endothelial growth factor (VEGF)-A, angiopoietin-1, angiopoietin-1/VEGF-A ratio, VEGF receptor 1 (VEGFR-1), VEGFR-2, and pentraxin-3 with CKD. METHODS: We recruited 201 patients with CKD and 201 community controls without CKD from the greater New Orleans area. CKD was defined as estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 or presence of albuminuria. Multivariable quantile and logistic regression models were used to examine the relationship between angiogenesis-related factors and CKD adjusting for confounding factors. RESULTS: After adjusting for covariables including traditional cardiovascular disease (CVD) risk factors, C-reactive protein, and history of CVD, the medians (interquartile range) were 133.08 (90.39, 204.15) in patients with CKD vs. 114.17 (72.45, 170.32) pg/mL in controls without CKD (p = 0.002 for group difference) for VEGF-A; 3951.2 (2471.9, 6656.6) vs. 4270.5 (2763.7, 6537.2) pg/mL (p = 0.70) for angiopoietin-1; 25.87 (18.09, 47.90) vs. 36.55 (25.71, 61.10) (p = 0.0001) for angiopoietin-1/VEGF-A ratio; 147.81 (122.94, 168.79) vs. 144.16 (123.74, 168.05) ng/mL (p = 0.25) for VEGFR-1; 26.20 (22.67, 29.92) vs. 26.28 (23.10, 29.69) ng/mL (p = 0.31) for VEGFR-2; and 1.01 (0.79, 1.49)vs. 0.89 (0.58, 1.18) ng/mL (p = 0.01) for pentraxin-3, respectively. In addition, an elevated VEGF-A level and decreased angiopoietin-1/VEGF-A ratio were associated with increased odds of CKD. CONCLUSIONS: These data indicate that plasma VEGF-A and pentraxin-3 levels were increased and the angiopoietin-1/VEGF-A ratio was decreased in patients with CKD. Future prospective studies are warranted to examine whether angiogenic factors play a role in progression of CKD.

Pharmaceutical Characterization of Tropomyosin Receptor Kinase B-Agonistic Antibodies on Human Induced Pluripotent Stem (hiPS) Cell-Derived Neurons.

Brain-derived neurotrophic factor (BDNF) is a central modulator of neuronal development and synaptic plasticity in the central nervous system. This renders the BDNF-modulated tropomyosin receptor kinase B (TrkB) a promising drug target to treat synaptic dysfunctions. Using GRowth factor-driven expansion and INhibition of NotCH (GRINCH) during maturation, the so-called GRINCH neurons were derived from human-induced pluripotent stem cells. These GRINCH neurons were used as model cells for pharmacologic profiling of two TrkB-agonistic antibodies, hereafter referred to as AB2 and AB20 In next-generation sequencing studies, AB2 and AB20 stimulated transcriptional changes, which extensively overlapped with BDNF-driven transcriptional modulation. In regard to TrkB phosphorylation, both AB2 and AB20 were only about half as efficacious as BDNF; however, with respect to the TrkB downstream signaling, AB2 and AB20 displayed increased efficacy values, providing a stimulation at least comparable to BDNF in respect to VGF transcription, as well as of AKT and cAMP response element-binding protein phosphorylation. In a complex structure of the TrkB-d5 domain with AB20, determined by X-ray crystallography, the AB20 binding site was found to be allosteric in regard to the BDNF binding site, whereas AB2 was known to act orthosterically with BDNF. In agreement with this finding, AB2 and AB20 acted synergistically at greater concentrations to drive TrkB phosphorylation. Although TrkB downstream signaling declined faster after pulse stimulation with AB20 than with AB2, AB20 restimulated TrkB phosphorylation more efficiently than AB2. In conclusion, both antibodies displayed some limitations and some benefits in regard to future applications as therapeutic agents.

Multidimensional gene search with Genehopper.

The high abundance of genetic information enables researchers to gain new insights from the comparison of human genes according to their similarities. However, existing tools that allow the exploration of such gene-to-gene relationships, apply each similarity independently. To make use of multidimensional scoring, we developed a new search engine named Genehopper. It can handle two query types: (i) the typical use case starts with a term-to-gene search, i.e. an optimized full-text search for an anchor gene of interest. The web-interface can handle one or more terms including gene symbols and identifiers of Ensembl, UniProt, EntrezGene and RefSeq. (ii) When the anchor gene is defined, the user can explore its neighborhood by a gene-to-gene search as the weighted sum of nine normalized gene similarities based on sequence homology, protein domains, mRNA expression profiles, Gene Ontology Annotation, gene symbols and other features. Each weight can be adjusted by the user, allowing flexible customization of the gene search. All implemented similarities have a low pairwise correlation (max r(2) = 0.4) implying a low linear dependency, i.e. any change in a single weight has an effect on the ranking. Thus, we treated them as separate dimensions in the search space. Genehopper is freely available at http://genehopper.ifis.cs.tu-bs.de.

Process-oriented dynamic group psychotherapy for depression as a teaching modality in a family medicine residency program- A pilot study.

Background and Objectives This pilot study provides a description and evaluation of process-oriented dynamic group psychotherapy for depression as a teaching modality for family medicine residents. The main purpose of using this modality was to teach family medicine residents a variety of psychological clinical skills. A secondary benefit of this modality was to provide in-house, primary care treatment to depressed patients, although the efficacy of this was not evaluated in the present study. Methods A 10-item, self-report, Likert-type questionnaire was administered to a convenience sample of family medicine residents who had participated in the program. Results Completed questionnaires were received from 100% of the family medicine resident participants. Responses to the questionnaires indicate that the residents felt they acquired a variety of clinical skills from the training modality, to include developing active listening and interviewing skills; methods to improve the doctor-patient relationship; increased skills in empathy, intuitive processes, and emotional support; a depth understanding of how intra-psychic conflicts and interpersonal problems contribute to depression; how to give effective feedback that promotes behavioral change; and how to place interventions at the appropriate level of change. Eighty-eight percent of residents indicated they would recommend this learning modality to a family medicine physician colleague. Conclusions The family medicine residents' responses to the questionnaires indicate that they perceived process-oriented dynamic group psychotherapy for depression as a constructive and beneficial modality for both patient care and learning a variety of clinical skills.

Programmed Cell Death Genes Are Linked to Elevated Creatine Kinase Levels in Unhealthy Male Nonagenarians.

Declining health in the oldest-old takes an energy toll for the simple maintenance of body functions. The underlying mechanisms, however, differ in males and females. In females, the declines are explained by loss of muscle mass; but this is not the case in males, in whom they are associated with increased levels of circulating creatine kinase. This relationship raises the possibility that muscle damage rather than muscle loss is the cause of the increased energy demands of unhealthy aging in males. We have now examined factors that contribute to the increase in creatine kinase. Much of it (60%) can be explained by a history of cardiac problems and lower kidney function, while being mitigated by moderate physical activity, reinforcing the notion that tissue damage is a likely source. In a search for genetic risk factors associated with elevated creatine kinase, the Ku70 gene XRCC6 and the ceramide synthase gene LASS1 were investigated because of their roles in telomere length and longevity and healthy aging, respectively. Single nucleotide polymorphisms in these two genes were independently associated with creatine kinase levels. The XRCC6 variant was epistatic to one of the LASS1 variants but not to the other. These gene variants have potential regulatory activity. Ku70 is an inhibitor of the proapoptotic Bax, while the product of Lass1, ceramide, operates in both caspase-dependent and -independent pathways of programmed cell death, providing a potential cellular mechanism for the effects of these genes on tissue damage and circulating creatine kinase.

Association of C-reactive protein, tumor necrosis factor-alpha, and interleukin-6 with chronic kidney disease.

BACKGROUND: We studied the association of inflammatory biomarkers including C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) with chronic kidney disease (CKD). METHODS: We conducted a case-control study among 201 CKD patients and 201 community-based controls in the greater New Orleans area. CKD was defined as estimated-glomerular filtration rate (eGFR) <60 mL/min/1.73 m(2) or albuminuria ≥30 mg/24-h. Serum CRP, TNF-α, and IL-6 were measured using standard methods. Multivariable regression models were used to examine associations between the inflammatory biomarkers and CKD adjusting for important CKD risk factors, history of cardiovascular disease, and use of antihypertensive, antidiabetic, and lipid-lowering agents and aspirin. RESULTS: The multivariable-adjusted medians (interquartile-range) were 2.91 (1.47, 5.24) mg/L in patients with CKD vs. 1.91 (0.99, 3.79) mg/L in controls without CKD (p = 0.39 for group difference) for CRP; 1.86 (1.51, 2.63) pg/mL vs. 1.26 (1.01, 1.98) pg/mL (p < 0.0001) for TNF-α; and 2.53 (1.49, 4.42) pg/mL vs. 1.39 (0.95, 2.15) pg/mL (p = 0.04) for IL-6, respectively. Compared to the lowest tertile, the highest tertile of TNF-α (OR 7.1, 95% CI 3.2 to 15.5) and IL-6 (OR 2.5, 95% CI 1.1 to 5.5) were significantly associated with higher odds of CKD in multivariable-adjusted models. Additionally, higher TNF-α and IL-6 were independently and significantly associated with lower eGFR and higher albuminuria. CONCLUSIONS: Our data suggest that TNF-α and IL-6, but not CRP, are associated with the prevalence and severity of CKD, independent from established CKD risk factors, history of cardiovascular disease, and use of antihypertensive, antidiabetic, and lipid-lowering agents and aspirin.

Diverse potential of secretome from natural killer cells and monocyte-derived macrophages in activating stellate cells.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-ß1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

Transcriptomic signatures of progressive and regressive liver fibrosis and portal hypertension.

Persistent liver injury triggers a fibrogenic program that causes pathologic remodeling of the hepatic microenvironment (i.e., liver fibrosis) and portal hypertension. The dynamics of gene regulation during liver disease progression and early regression remain understudied. Here, we generated hepatic transcriptome profiles in two well-established liver disease models at peak fibrosis and during spontaneous regression after the removal of the inducing agents. We linked the dynamics of key disease readouts, such as portal pressure, collagen area, and transaminase levels, to differentially expressed genes, enabling the identification of transcriptomic signatures of progressive vs. regressive liver fibrosis and portal hypertension. These candidate biomarkers (e.g., Tcf4, Mmp7, Trem2, Spp1, Scube1, Islr) were validated in RNA sequencing datasets of patients with cirrhosis and portal hypertension, and those cured from hepatitis C infection. Finally, deconvolution identified major cell types and suggested an association of macrophage and portal hepatocyte signatures with portal hypertension and fibrosis area.

An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Urine free light chains as a novel biomarker of acute kidney allograft injury.

BACKGROUND: We evaluated urine free light chains (FLC) as a potential biomarker for acute kidney allograft injury (AKAI). METHODS: Urine κ and λ FLC were compared with urine ß-2 microglobulin (ß2-M), retinol-binding protein (RBP), kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and microalbuminuria (MAB) in biopsy-confirmed acute rejection (AR) and acute tubular necrosis (ATN). Healthy volunteers (normal) and transplant recipients with normal allograft function (control) were used as references. RESULTS: Compared with control or normal group (N = 15), urine FLC, MAB, and RBP were higher in ATN (N = 29) and AR (N = 41) groups (p < 0.05). There was no difference in KIM-1, NGAL, or ß2-M between four groups. In the AR group, urine κFLC demonstrated the highest predictive value with sensitivity of 95.12% and specificity of 87.5% (p < 0.0001). Urine κFLC also performed best with a sensitivity of 96.55% and specificity of 93.33% (p < 0.0001) in the ATN group. The area under the receiver operating characteristic (ROC) curves (AUC) by ROC analysis is greatest in urine RBP (100%) and FLC (99%), and lowest in KIM-1 (53.5%), then NGAL (71.5%) in the AR group. The AUC is also greatest in urine FLC (100%) and RBP (99%), and lowest in urine KIM-1 (55.6%) and NGAL (69.9%) in the ATN group. CONCLUSIONS: Urine FLC appears sensitive for both AR and ATN, and it may be a novel AKAI biomarker.

Early inhibition of the renin-angiotensin system improves the long-term graft survival of single pediatric donor kidneys transplanted in adult recipients.

Transplanting single pediatric donor kidneys into adult recipients has an increased risk of hyperfiltration injury and graft loss. It is unknown if renin-angiotensin system (RAS) blockers are beneficial in this setting. We retrospectively analyzed 94 adults who received single kidneys from donors <10 years old during 1996-2009. The recipients were divided into group 1 with RAS blockers (n = 40) and group 2 without RAS blockers (n = 54) in the first year of transplant. There was no significant difference in any donor/recipient demographic between the two groups. Graft function, incidence of delayed graft function, acute rejection, and persistent proteinuria were not statistically different either. Kaplan-Meier estimated death-censored graft survivals were significantly better in group 1 than in group 2: 95 vs. 81.2%, 82.4 vs. 61.2%, 72.6 vs. 58.5%, and 68.5 vs. 47.2% at 1, 3, 5, and 7 years, respectively (log rank P = 0.043). Multivariable analysis found persistent proteinuria was a risk factor for graft loss (OR 2.70, 95% CI 1.33-5.49, P = 0.006), while RAS blockers reduced the risk of graft loss (OR 0.38, 95% CI 0.18-0.79, P = 0.009). Early RAS blockade therapy in the first year of transplant is associated with superior long-term graft survival among adults transplanted with single pediatric donor kidneys.