Single-Component Multilayered Self-Assembling Protein Nanoparticles Displaying Extracellular Domains of Matrix Protein 2 as a Pan-influenza A Vaccine.

The development of a cross-protective pan-influenza A vaccine remains a significant challenge. In this study, we designed and evaluated single-component self-assembling protein nanoparticles (SApNPs) presenting the conserved extracellular domain of matrix protein 2 (M2e) as vaccine candidates against influenza A viruses. The SApNP-based vaccine strategy was first validated for human M2e (hM2e) and then applied to tandem repeats of M2e from human, avian, and swine hosts (M2ex3). Vaccination with M2ex3 displayed on SApNPs demonstrated higher survival rates and less weight loss compared to the soluble M2ex3 antigen against the lethal challenges of H1N1 and H3N2 in mice. M2ex3 I3-01v9a SApNPs formulated with a squalene-based adjuvant were retained in the lymph node follicles over 8 weeks and induced long-lived germinal center reactions. Notably, a single low dose of M2ex3 I3-01v9a SApNP formulated with a potent adjuvant, either a Toll-like receptor 9 (TLR9) agonist or a stimulator of interferon genes (STING) agonist, conferred 90% protection against a lethal H1N1 challenge in mice. With the ability to induce robust and durable M2e-specific functional antibody and T cell responses, the M2ex3-presenting I3-01v9a SApNP provides a promising pan-influenza A vaccine candidate.

Discriminatory Ability and Clinical Utility of the AJCC7 and AJCC8 Staging Systems for Breast Cancer in a Middle-Income Setting.

(1) Background: Differences in access to biomarker testing and cancer treatment in resource-limited settings may affect the clinical utility of the AJCC8 staging system compared to the anatomical AJCC7 system. (2) Methods: A total of 4151 Malaysian women who were newly diagnosed with breast cancer from 2010 to 2020 were followed-up until December 2021. All patients were staged using the AJCC7 and AJCC8 systems. Overall survival (OS) and relative survival (RS) were determined. Concordance-index was used to compare the discriminatory ability between the two systems. (3) Results: Migration from the AJCC7 to AJCC8 staging system resulted in the downstaging of 1494 (36.0%) patients and the upstaging of 289 (7.0%) patients. Approximately 5% of patients could not be staged using the AJCC8 classification. Five-year OS varied between 97% (Stage IA) and 66% (Stage IIIC) for AJCC7, and 96% (Stage IA) and 60% (Stage IIIC) for AJCC8. Concordance-indexes for predicting OS using the AJCC7 and AJCC8 models were 0.720 (0.694-0.747) and 0.745 (0.716-0.774), and for predicting RS they were 0.692 (0.658-0.728) and 0.710 (0.674-0.748), respectively. (4) Conclusions: Given the comparable discriminatory ability between the two staging systems in predicting the stage-specific survival of women with breast cancer in the current study, the continued use of the AJCC7 staging system in resource-limited settings seems pragmatic and justifiable.

Adherence trajectory as an on-treatment risk indicator among drug-resistant TB patients in the Philippines.

INTRODUCTION: High levels of treatment adherence are critical for achieving optimal treatment outcomes among patients with tuberculosis (TB), especially for drug-resistant TB (DR TB). Current tools for identifying high-risk non-adherence are insufficient. Here, we apply trajectory analysis to characterize adherence behavior early in DR TB treatment and assess whether these patterns predict treatment outcomes. METHODS: We conducted a retrospective analysis of Philippines DR TB patients treated between 2013 and 2016. To identify unique patterns of adherence, we performed group-based trajectory modelling on adherence to the first 12 weeks of treatment. We estimated the association of adherence trajectory group with six-month and final treatment outcomes using univariable and multivariable logistic regression. We also estimated and compared the predictive accuracy of adherence trajectory group and a binary adherence threshold for treatment outcomes. RESULTS: Of 596 patients, 302 (50.7%) had multidrug resistant TB, 11 (1.8%) extremely drug-resistant (XDR) TB, and 283 (47.5%) pre-XDR TB. We identified three distinct adherence trajectories during the first 12 weeks of treatment: a high adherence group (n = 483), a moderate adherence group (n = 93) and a low adherence group (n = 20). Similar patterns were identified at 4 and 8 weeks. Being in the 12-week moderate or low adherence group was associated with unfavorable six-month (adjusted OR [aOR] 3.42, 95% CI 1.90-6.12) and final (aOR 2.71, 95% 1.73-4.30) treatment outcomes. Adherence trajectory group performed similarly to a binary threshold classification for the prediction of final treatment outcomes (65.9% vs. 65.4% correctly classified), but was more accurate for prediction of six-month treatment outcomes (79.4% vs. 60.0% correctly classified). CONCLUSIONS: Adherence patterns are strongly predictive of DR TB treatment outcomes. Trajectory-based analyses represent an exciting avenue of research into TB patient adherence behavior seeking to inform interventions which rapidly identify and support patients with high-risk adherence patterns.

Examining thein vivofunctionality of the magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI).

Objective. Although neural-enabled prostheses have been used to restore some lost functionality in clinical trials, they have faced difficulty in achieving high degree of freedom, natural use compared to healthy limbs. This study investigated thein vivofunctionality of a flexible and scalable regenerative peripheral-nerve interface suspended within a microchannel-embedded, tissue-engineered hydrogel (the magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI)) as a potential approach to improving current issues in peripheral nerve interfaces.Approach. Assembled MARTEENI devices were implanted in the gaps of severed sciatic nerves in Lewis rats. Both acute and chronic electrophysiology were recorded, and channel-isolated activity was examined. In terminal experiments, evoked activity during paw compression and stimulus response curves generated from proximal nerve stimulation were examined. Electrochemical impedance spectroscopy was performed to assess the complex impedance of recording sites during chronic data collection. Features of the foreign-body response (FBR) in non-functional implants were examined using immunohistological methods.Main results. Channel-isolated activity was observed in acute, chronic, and terminal experiments and showed a typically biphasic morphology with peak-to-peak amplitudes varying between 50 and 500µV. For chronic experiments, electrophysiology was observed for 77 days post-implant. Within the templated hydrogel, regenerating axons formed minifascicles that varied in both size and axon count and were also found to surround device threads. No axons were found to penetrate the FBR. Together these results suggest the MARTEENI is a promising approach for interfacing with peripheral nerves.Significance. Findings demonstrate a high likelihood that observed electrophysiological activity recorded from implanted MARTEENIs originated from neural tissue. The variation in minifascicle size seen histologically suggests that amplitude distributions observed in functional MARTEENIs may be due to a combination of individual axon and mini-compound action potentials. This study provided an assessment of a functional MARTEENI in anin vivoanimal model for the first time.

Electrochemical modulation enhances the selectivity of peripheral neurostimulation in vivo.

Electrical nerve stimulation serves an expanding list of clinical applications, but it faces persistent challenges in selectively activating bundled nerve fibers. In this study, we investigated electrochemical modulation with an ion-selective membrane (ISM) and whether it, used together with electrical stimulation, may provide an approach for selective control of peripheral nerves. Guided by theoretical transport modeling and direct concentration measurements, we developed an implantable, multimodal ISM cuff capable of simultaneous electrical stimulation and focused Ca2+ depletion. Acutely implanting it on the sciatic nerve of a rat in vivo, we demonstrated that Ca2+ depletion could increase the sensitivity of the nerve to electrical stimulation. Furthermore, we found evidence that the effect of ion modulation would selectively influence functional components of the nerve, allowing selective activation by electrical current. Our results raise possibilities for improving functional selectivity of new and existing bioelectronic therapies, such as vagus nerve stimulation.

Impact of shelter-in-place orders on TB case notifications and mortality in the Philippines during the COVID-19 pandemic.

BACKGROUND: Policies implemented to slow transmission of COVID-19 are expected to have disrupted delivery of routine health services, including tuberculosis (TB) care. METHODS: We analyzed daily counts of drug-susceptible (DS)-TB case notifications from all health facilities affiliated with the Philippines National TB Program (NTP) before and after implementation of community quarantine (January 1-December 31, 2020). Using an interrupted time series design, we assessed the immediate and sustained effects of community quarantine on TB case reporting. Using 2019 WHO estimates of national TB incidence, treatment, and mortality rates for the Philippines, we modeled excess mortality from TB, assuming a national decline in TB case reporting were extended for 12 months, followed by a return to pre-community quarantine trends. RESULTS: The analysis included 192,918 DS-TB case notifications from 2,986 facilities located in 113 provinces and highly urbanized cities across 17 regions and covered 49 observations days before and 174 days after community quarantine implementation. We found an significant drop and steeper decline in daily TB case notifications after the implementation of community quarantine, resulting in 44.6% (95% CI 38.3-50.1) fewer daily TB case notifications 60 days after implementation of community quarantine. During 2020, DS-TB case notifications never returned to pre-quarantine levels. Assuming a 12-month disruption of TB case reporting, we estimate there will be 56.3% increase in TB-related deaths in the Philippines. CONCLUSION: Modified delivery of TB prevention and care should be prioritized alongside efforts to combat COVID-19.

Rapid, label-free classification of tumor-reactive T cell killing with quantitative phase microscopy and machine learning.

Quantitative phase microscopy (QPM) enables studies of living biological systems without exogenous labels. To increase the utility of QPM, machine-learning methods have been adapted to extract additional information from the quantitative phase data. Previous QPM approaches focused on fluid flow systems or time-lapse images that provide high throughput data for cells at single time points, or of time-lapse images that require delayed post-experiment analyses, respectively. To date, QPM studies have not imaged specific cells over time with rapid, concurrent analyses during image acquisition. In order to study biological phenomena or cellular interactions over time, efficient time-dependent methods that automatically and rapidly identify events of interest are desirable. Here, we present an approach that combines QPM and machine learning to identify tumor-reactive T cell killing of adherent cancer cells rapidly, which could be used for identifying and isolating novel T cells and/or their T cell receptors for studies in cancer immunotherapy. We demonstrate the utility of this method by machine learning model training and validation studies using one melanoma-cognate T cell receptor model system, followed by high classification accuracy in identifying T cell killing in an additional, independent melanoma-cognate T cell receptor model system. This general approach could be useful for studying additional biological systems under label-free conditions over extended periods of examination.

Rapid and Low Cost Manufacturing of Cuff Electrodes.

For many peripheral neuro-modulation applications, the cuff electrode has become a preferred technology for delivering electrical current into targeted volumes of tissue. While basic cuffs with low spatial selectivity, having longitudinally arranged contacts, can be produced from relatively straightforward processes, the fabrication of more complex electrode configurations typically requires iterative design and clean-room fabrication with skilled technicians. Although facile methods for fabricating cuff electrodes exist, their inconsistent products have limited their adoption for rapid manufacturing. In this article, we report a fast, low-cost fabrication process for patterning of electrode contacts in an implantable peripheral nerve cuff. Using a laser cutter as we have prescribed, the designer can render precise contact geometries that are consistent between batches. This method is enabled by the use of silicone/carbon black (CB) composite electrodes, which integrate with the patterned surface of its substrate-tubular silicone insulation. The size and features of its products can be adapted to fit a wide range of nerve diameters and applications. In this study, we specifically documented the manufacturing and evaluation of circumpolar cuffs with radial arrays of three contacts for acute implantation on the rat sciatic nerve. As part of this method, we also detail protocols for verification-electrochemical characterization-and validation-electrophysiological evaluation-of implantable cuff electrodes. Applied to our circumpolar cuff electrode, we report favorable electrical characteristics. In addition, we report that it reproduces expected electrophysiological behaviors described in prior literature. No specialized equipment or fabrication experience was required in our production, and we encountered negligible costs relative to commercially available solutions. Since, as we demonstrate, this process generates consistent and precise electrode geometries, we propose that it has strong merits for use in rapid manufacturing.

Immunotherapy in Hepatocellular Carcinoma: Is There a Light at the End of the Tunnel?

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with dismal prognosis when diagnosed at advanced stages. Surgical resection of the primary tumor or orthotropic liver transplantation serves as a potential curative option. However, this approach is highly dependent on the hepatic reserve and baseline functional status of the patient. Liver directed therapies such as portal vein embolization (PVE), trans-arterial chemoembolization (TACE), and systemic chemotherapy are employed in non-surgical candidates. Sorafenib was the only approved systemic therapeutic agent for almost a decade until the recent approval of lenvatinib by the United States Food and Drug Administration (FDA) as an alternate first-line agent. Regorafenib, nivolumab, pembrolizumab and cabozantinib are approved by the FDA as second-line agents in patients who failed or could not tolerate sorafenib. Ramucirumab was recently FDA approved for the subset of patients that have high alfa-fetoprotein levels (>400 ng/mL). A better understanding of tumorigenesis and encouraging clinical trial results that evaluated immune-checkpoint inhibitors opened doors for immunotherapy in HCC. Immune checkpoint inhibitors have demonstrated a prolonged median overall and progression-free survival in a subset of patients with HCC. On-going translational and clinical research will hopefully provide us with a better understanding of tumor markers, genetic aberrations and other factors that determine the immunotherapy response in HCC. In this review, we sought to summarize the potential role and future directions of immunotherapy in the management of HCC.

Pan-Genomic and Polymorphic Driven Prediction of Antibiotic Resistance in Elizabethkingia.

The Elizabethkingia are a genetically diverse genus of emerging pathogens that exhibit multidrug resistance to a range of common antibiotics. Two representative species, Elizabethkingia bruuniana and E. meningoseptica, were phenotypically tested to determine minimum inhibitory concentrations (MICs) for five antibiotics. Ultra-long read sequencing with Oxford Nanopore Technologies (ONT) and subsequent de novo assembly produced complete, gapless circular genomes for each strain. Alignment based annotation with Prokka identified 5,480 features in E. bruuniana and 5,203 features in E. meningoseptica, where none of these identified genes or gene combinations corresponded to observed phenotypic resistance values. Pan-genomic analysis, performed with an additional 19 Elizabethkingia strains, identified a core-genome size of 2,658,537 bp, 32 uniquely identifiable intrinsic chromosomal antibiotic resistance core-genes and 77 antibiotic resistance pan-genes. Using core-SNPs and pan-genes in combination with six machine learning (ML) algorithms, binary classification of clindamycin and vancomycin resistance achieved f1 scores of 0.94 and 0.84, respectively. Performance on the more challenging multiclass problem for fusidic acid, rifampin and ciprofloxacin resulted in f1 scores of 0.70, 0.75, and 0.54, respectively. By producing two sets of quality biological predictors, pan-genome genes and core-genome SNPs, from long-read sequence data and applying an ensemble of ML techniques, our results demonstrated that accurate phenotypic inference, at multiple AMR resolutions, can be achieved.

Nanopore ultra-long read sequencing technology for antimicrobial resistance detection in Mannheimia haemolytica.

Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were sequenced at 146× and 111× coverage, respectively with Oxford Nanopore Technologies MinION. De novo assembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including ß-lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Resistance phenotypes of the M. haemolytica strains were determined by minimum inhibitory concentration (MIC) of the antibiotics. Sequencing with a highly portable MinION device corresponded to MIC assays with most of the antimicrobial resistant determinants being identified with as few as 5437 reads, except for the genes responsible for resistance to Fluoroquinolones. The resulting quality assemblies and AMR gene annotation highlight the efficiency of ultra-long read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

Novel targeted treatment options for advanced cholangiocarcinoma.

INTRODUCTION: Surgical resection remains the mainstay of potentially curative treatment in the early stages of cholangiocarcinoma, whereas for the advanced stage, systemic chemotherapeutics and experimental targeted therapies are the primary treatment options. The molecular heterogeneity of the tumor is based on location, liver dysfunction, and relative rarity of the disease and confers challenges for clinical trial enrollment. The advancements in the understanding of molecular pathogenesis of cholangiocarcinoma have led to the development of targeted therapies that are currently being evaluated in the clinical trials. AREAS COVERED: This review summarizes the current understanding and future directions of targeted therapeutic options in the management of advanced cholangiocarcinoma. EXPERT OPINION: Advanced cholangiocarcinoma has a dismal prognosis; improved understanding of the molecular pathogenesis and advancements in development of targeted therapy offers hope that we may improve outcomes in this rare, but highly lethal cancer. Among the newly discovered molecular alterations, targeting FGFR2 fusions, IDH1/2 mutations and HER2 receptors hold great promise for improving the future management of cholangiocarcinoma. Immunotherapy in combination with targeted agents and chemotherapy may improve outcomes. In addition, drugs targeting the MEK, EGFR, KRAS, BRAF, and ROS1 pathways and neo-angiogenesis may also provide new horizons in the management of cholangiocarcinoma.

Diagnosis and Monitoring of Prostatic Lesions: A Comparison of Three Modalities: Multiparametric MRI, Fusion MRI/Transrectal Ultrasound (TRUS), and Traditional TRUS.

PURPOSE:  Transrectal ultrasound (TRUS) has been the gold standard of imaging for diagnosing prostate cancer for decades but is plagued by user error and undersampling. We aim to explore imaging modalities that are now being used in combination or alone for screening, diagnosis, and/or active surveillance of prostate cancer. METHODS: A PubMed literature search was performed to include articles published up to April 2016. Data were extracted and analyzed. RESULTS: Several large-scale studies have found an increased cancer detection rate in MRI-targeted lesions with an improved ability to target anterior lesions as well as an increased cancer detection in high-risk cancers using fusion platforms vs TRUS alone. CONCLUSIONS: To date, there have been few head-to-head trials to directly compare the use of multiparametric MRI (mpMRI), transrectal ultrasound, and MRI-ultrasound fusion modalities for accurate and reliable detection, active surveillance, or biopsy procedure success rates. Further investigation utilizing these modalities are needed before they can be relied upon in active surveillance management, although mpMRI appears to be currently the most reliable in monitoring and diagnosing prostate lesions.

Isolation and cultivation of human articular chondrocytes.

Adult human articular chondrocytes were isolated from surgically excised articular cartilage from 15 patients suffering from trauma or disease of the knee. Cells were isolated with an enzymatic digestion method and cultured with F12 medium supplemented with serum. The effects of serum and basic fibroblast growth factor (bFGF) on the growth of cultured chondrocytes were studied. Serum stimulated the growth of chondrocytes at concentrations from 1-30%. bFGF significantly stimulated the growth of chondrocytes in a dose-dependent manner at concentrations from 1-100 ng/ml. Chondrocytes grew well in F12 medium supplemented with 10% fetal bovine serum (FBS). These cultured chondrocytes could be passaged for many generations and revealed an average of 2,702-fold increase of cell numbers during 2-6 months period (cumulative population doublings = 11 times). Cumulative population doublings increased to 24 times in cell cultured with medium supplemented with bFGF (10 ng/ml). Immunocytochemical study using anti-S-100 antibodies demonstrated that these cultures were pure cell cultures of chondrocytes. We have demonstrated that cell cultures of adult human articular chondrocytes can be established with these methods. Cultured chondrocytes provide an in vitro model system for studying the physiology and pathology of human articular chondrocytes and may be used for autologous transplantation of chondrocytes to treat articular cartilage defects.