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Purpose: Hyperglycemia is a major risk factor for early lesions of diabetic retinal disease (DRD). Updating the DRD staging system to incorporate relevant basic and cellular mechanisms pertinent to DRD is necessary to better address early disease, disease progression, the use of therapeutic interventions, and treatment effectiveness. Design: We sought to review preclinical and clinical evidence on basic and cellular mechanisms potentially pertinent to DRD that might eventually be relevant to update the DRD staging system. Participants: Not applicable. Methods: The Basic and Cellular Mechanisms Working Group (BCM-WG) of the Mary Tyler Moore Vision Initiative carefully and extensively reviewed available preclinical and clinical evidence through multiple iterations and classified these. Main Outcome Measures: Classification was made into evidence grids, level of supporting evidence, and anticipated future relevance to DRD. Results: A total of 40 identified targets based on pathophysiology and other parameters for DRD were grouped into concepts or evaluated as specific candidates. VEGFA, peroxisome proliferator-activated receptor-alpha related pathways, plasma kallikrein, and angiopoietin 2 had strong agreement as promising for use as biomarkers in diagnostic, monitoring, predictive, prognostic, and pharmacodynamic responses as well as for susceptibility/risk biomarkers that could underlie new assessments and eventually be considered within an updated DRD staging system or treatment, based on the evidence and need for research that would fit within a 2-year timeline. The BCM-WG found there was strong reason also to pursue the following important concepts regarding scientific research of DRD acknowledging their regulation by hyperglycemia: inflammatory/cytokines, oxidative signaling, vasoprotection, neuroprotection, mitophagy, and nutrients/microbiome. Conclusion: Promising targets that might eventually be considered within an updated DRD staging system or treatment were identified. Although the BCM-WG recognizes that at this stage little can be incorporated into a new DRD staging system, numerous potential targets and important concepts deserve continued support and research, as they may eventually serve as biomarkers and/or therapeutic targets with measurable benefits to patients with diabetes. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Summary: Background: International distribution of contaminated foods can be a source of Salmonella infections in people and can contribute to the spread of antimicrobial-resistant bacteria across countries. We report an investigation led by the United States Centers for Disease Control and Prevention, the Food and Drug Administration (FDA), and state governmental officials into a multistate outbreak of salmonellosis linked to pig ear pet treats. Methods: Pig ear treats and companion dogs were tested for Salmonella by state officials and the FDA. Products were traced back to the country of origin when possible. Cases were defined as outbreak illnesses in people associated with one of seven Salmonella serotypes genetically related to samples from pig ear pet treats, with isolation dates from June 2015 to September 2019. Whole genome sequencing (WGS) of isolates was used to predict antimicrobial resistance. Findings: The outbreak included 154 human cases in 34 states. Of these, 107 of 122 (88%) patients reported dog contact, and 65 of 97 (67%) reported contact with pig ear pet treats. Salmonella was isolated from 137 pig ear treats, including some imported from Argentina, Brazil, and Colombia, and from four dogs. WGS predicted 77% (105/137) of human and 43% (58/135) of pig ear treat isolates were resistant to ≥3 antimicrobial classes. Interpretation: This was the first documented United States multistate outbreak of Salmonella infections linked to pig ear pet treats. This multidrug-resistant outbreak highlights the interconnectedness of human health and companion animal ownership and the need for zoonotic pathogen surveillance to prevent human illness resulting from internationally transported pet food products. Funding: Animal Feed Regulatory Program Standards award. Animal and product testing conducted by FDA Vet-LIRN was funded by Vet-LIRN infrastructure grants (PAR-22-063).

ABSTRACT Objective: To evaluate the newborn hearing screening program in the University of Santo Tomas Hospital based on the quality indicators set by the Joint Committee on Infant Hearing (JCIH) 2019 position statement. Methods: Design: Cross-sectional study Setting: Tertiary Private Training Hospital Participants: All newborns delivered in 2019 at the University of Santo Tomas (UST) Hospital were considered for inclusion Results: The UST Hospital had 778 newborns in 2019, of which 687 (88.3%) completed newborn hearing screening by 1 month of age. There were 81 (10.4%) who failed initial hospitalbased screening and required outpatient re-screening while 11 (1.4%) of those who failed initial screening also failed subsequent rescreening. Forty-five (5.7%) newborns failed initial screening and subsequently passed re-screening. None of the eleven (0/11) patients completed comprehensive audiologic evaluation thus, patients necessitating referral for intervention were not identified. There were a total of 67 (8.6%) dropouts throughout the hearing evaluation process. Fifteen (15) infants were not screened due to unavailability of trained personnel at time of referral, four (4) infants were advised third screening while two (2) were advised observation instead of proceeding to confirmatory test. Conclusion: The University of Santo Tomas Hospital newborn hearing screening program has yet to reach the quality indicators set by the Joint Committee on Infant Hearing 2019 for screening and confirmation of hearing loss. Among identified areas for improvement are the availability of trained personnel, insufficient means to ensure compliance, reluctance to pursue further testing and practices among healthcare providers

Background Unplanned intensive care unit (ICU) readmission—ICU bounce back (ICUbb)—is associated with worse outcomes. Patients not requiring organ system support or intensive nursing are deemed ‘ICU discharge ready’ and transfer orders are placed. However, actual transfer only occurs when an appropriate, non-ICU bed is available. This is dependent on inherent system inefficiencies resulting in a naturally controlled experiment between when patients actually transfer: Early (24 hours) transfers, after order placement. This study leverages that natural experiment to determine if additional ICU time is protective against ICUbb. We hypothesize that Delayed transfer is protective against ICUbb.Methods Using a retrospective, cohort design, we queried a trauma research repository and electronic medical record during a 10-year period to capture traumatized patients admitted to the ICU. Patients were categorized into Early (24 hours) groups based on actual transfer time after order placement. Patient characteristics (age, Charlson Comorbidity Index (CCI)) and Injury Severity Score (ISS) were analyzed. Univariate and multivariate analyses were performed to compare ICUbb rates among Early and Unintended-Delayed groups.Results Of the 2004 patients who met the criteria, 1690 fell into the Early group, and 314 fell into the Delayed. The Early group was younger (mean age 52±23 vs. 55±22 years), had fewer comorbidities (median CCI score 1 (0, 3) vs. 2 (1, 3)), and was less injured (median ISS 17 (10–22) vs. 17 (13–25)), all p

Hamstring strains are the most common injury in elite football and typically occur during high-speed running. Despite its important contribution to power production in the late swing phase, injury to the semimembranosus (SM) is less common than to the biceps femoris, but may involve the free tendon and depending on the degree of retraction, warrant surgical repair. Few case reports detail clinical reasoning, supported by objective data during rehabilitation in elite footballers, and none have described the return to sport (RTS) process following this type of hamstring injury. In this article, we outline the management and RTS of an English Premier League (EPL) footballer who suffered a high-grade SM proximal tendon tear during training. Due to the degree of retraction of the free tendon, the player underwent surgical reconstruction at the recommendation of an orthopaedic surgeon. Early physiotherapy care, nutritional support, on- and off-pitch injury-specific reconditioning and global athletic development are outlined, alongside strength and power diagnostic and global positioning systems data, assessment of pain, player feedback and MRI informed clinical reasoning and shared decision-making during the RTS process. 18 weeks post-surgery the player returned to team training, transferring to a new club 3 weeks later. 2.5 years post RTS, the player remains free of re-injury playing regularly in the EPL.

Purpose. To characterize hallmark diabetic retinopathy (DR) lesions utilizing adaptive optics scanning laser ophthalmoscopy (AOSLO) and to compare AOSLO findings with those on standard imaging techniques. Methods. Cross-sectional study including 35 eyes of 34 study participants. AOSLO confocal and multiply scattered light (MSL) imaging were performed in eyes with DR. Color fundus photographs (CF), infrared images of the macula (Spectralis, Heidelberg), and Spectralis spectral domain optical coherence tomography SDOCT B-scans of each lesion were obtained and registered to corresponding AOSLO images. Main Outcome Measures. Individual lesion characterization by AOSLO imaging. AOSLO appearance was compared with CF and SDOCT imaging. Results. Characterized lesions encompassed 52 microaneurysms (MA), 20 intraretinal microvascular abnormalities (IRMA), 7 neovascularization (NV), 11 hard exudates (HE), 5 dot/blot hemorrhages (HEM), 4 cotton wool spots (CWS), and 14 intraretinal cysts. AOSLO allowed assessment of perfusion in vascular lesions and enabled the identification of vascular lesions that could not be visualized on CF or SDOCT. Conclusions. AOSLO imaging provides detailed, noninvasive in vivo visualization of DR lesions enhancing the assessment of morphological characteristics. These unique AOSLO attributes may enable new insights into the pathological changes of DR in response to disease onset, development, regression, and response to therapy.

Objective: To report on the implementation and initial pilot data of diabetic retinopathy (DR) identified using a validated telemedicine program for DR in a multispecialty primary care clinic. Methods: This was a retrospective, cross-sectional pilot survey of diabetic patients imaged for the first time at the telemedicine program of The Medical City from November 26, 2012 to August 31, 2013. The retinal images were obtained following the validated program of the Joslin Vision Network nonmydriatic fundus photography for DR evaluation. These were evaluated by 4 retina specialists using a standardized protocol to assess for diabetic retinal disease and the presence of other retinal findings. The findings were recorded on customized electronic templates. Results: Seven hundred seventy-six (776) eyes of 388 patients were evaluated using the telemedicine program. The prevalence of DR was 28.2% (219 eyes), with 25.1% (195 eyes) having nonproliferative DR (NPDR). 14.2% [110 eyes] had mild, 8.2% [64 eyes] moderate, 2.2% [17 eyes] severe, and 0.5% [4 eyes] very severe NPDR. 3.1% (24 eyes) had proliferative DR (PDR), of which 45.8% (11 eyes) had high-risk characteristics. The ungradable rate with selective mydriasis was 1.80% (14 eyes). The rate of referable DR (moderate NPDR or worse, any level of diabetic macular edema, or ungradable images) was 21.90% (170 eyes). Conclusion: In the primary care setting, teleophthalmology can effectively identify patients with diabetic retinal complications and potentially refer these patients to appropriate levels of eye care. Retinopathy was present in over 28% of patients evaluated and over 21% had referable disease that may potentially progress to vision loss. Teleophthalmology for DR in this setting allows early detection of potentially sight threatening disease and may prevent visual loss and complications.

More than half-a-billion individuals are projected to have diabetes by 2030, with the greatest increase in developing nations. Southeast Asian countries comprise a substantial proportion of this population and will need to prepare for both the rise in diabetes cases and the accompanying increase in diabetes related complications, most commonly diabetic retinopathy. Ocular telehealth programs utilizing retinal imaging for diabetic retinopathy have been established as an effective means to prevent visual loss due to diabetic retinal disease across multiple populations and in various settings. An additional value of ocular telehealth programs is their ability to integrate a multidisciplinary approach to diabetes eye care that incorporates patient education and fosters collaboration among multiple specialties. A program that provides a systematic and organized approach in delivering diabetes eye care is critically needed to successfully address this critical emerging health issue in this region of the world.

Reproductive outcomes, such as preterm birth, miscarriage/stillbirth, and pre-eclampsia, are understudied in veterans, particularly among Gulf War veterans (GWVs). During deployment, women GWVs were exposed to toxicant and nontoxicant exposures that may be associated with adverse reproductive and developmental outcomes. The data come from a survey of 239 participants from northeastern and southern U.S. cohorts of women veterans. The questionnaire collected information about the service history, current and past general health, reproductive and family health, demographic information, and deployment exposures. Odds ratios were computed with 95% confidence intervals between exposures in theater and reproductive/children's health outcomes. GWVs experienced adverse reproductive outcomes: 25% had difficulty conceiving, and 31% had a pregnancy that ended in a miscarriage or stillbirth. Pregnancy complications were common among GWVs: 23% had a high-risk pregnancy, and 16% were diagnosed with pre-eclampsia. About a third of GWVs reported their children (38%) had a developmental disorder. Use of pesticide cream during deployment was associated with higher odds of all reproductive and developmental outcomes. The results demonstrate that GWVs experienced reproductive and children's health outcomes at potentially high rates, and exploratory analyses suggest pesticide exposure as associated with higher odds of adverse reproductive outcomes. Future longitudinal studies of women veterans should prioritize examining reproductive and children's health outcomes.

Prevalence of nine chronic medical conditions in the population-based Ft. Devens Cohort (FDC) of GW veterans were compared with the population-based 2013⁻2014 National Health and Nutrition Examination Survey (NHANES) cohort. Excess prevalence was calculated as the difference in prevalence estimates from the Ft. Devens and NHANES cohorts; and confidence intervals and p -values are based on the standard errors for the two prevalence estimates. FDC males were at increased risk for reporting seven chronic medical conditions compared with NHANES males. FDC females were at decreased risk for high blood pressure and increased risk for diabetes when compared with NHANES females. FDC veterans reporting war-related chemical weapons exposure showed higher risk of high blood pressure; diabetes; arthritis and chronic bronchitis while those reporting taking anti-nerve gas pills had increased risk of heart attack and diabetes. GW veterans are at higher risk of chronic conditions than the general population and these risks are associated with self-reported toxicant exposures.

Purpose: This paper reports on a phenomenological study of women's experiences 1-10 years following a radical vaginal trachelectomy and describes the impact on health, sexuality, fertility and perceived supportive care needs., Method and Sample: Qualitative telephone interviews employing a descriptive phenomenological approach were conducted using a purposive sample of 12 women., Key Results: Several felt their cancer experience was positive; bringing them closer to family and changed their outlook on life. A few experienced delayed psychological reactions and/or fears of recurrence. Many experienced isolation and the desire to contact others with similar experiences. Women recovered well but a few experienced fears/concerns about lymphoedema and intermenstrual bleeding. Sexual function was not a long-term issue for most. Some that could feel the cerclage (stitch) during intercourse, developed techniques to reduce this. Single women felt vulnerable in new relationships. Pregnancy was an anxious time, especially for those that experienced a miscarriage or pre-term birth. Sources of support included the clinical nurse specialist, family/friends, surgical consultant, online patient forums and a support group. Women needed more information on trachelectomy statistics, pregnancy care recommendations as well as access to counselling, peer support, being seen by the same person and increased public awareness., Conclusions: This study has provided an interesting and detailed insight into women's experiences in the years following a trachelectomy, with results that have important considerations for practice such as provision of statistical information; counselling; peer support; consistent pregnancy recommendations; increased public awareness and increased identification and management or prevention of long-term physical effects., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The PPPY motif in the matrix (MA) domain of human T-cell leukemia virus type 1 (HTLV-1) Gag associates with WWP1, a member of the HECT domain containing family of E3 ubiquitin ligases. Mutation of the PPPY motif arrests particle assembly at an early stage and abolishes ubiquitination of MA. Similar effects are seen when Gag is expressed in the presence of a truncated form of WWP1 that lacks the catalytically active HECT domain (C2WW). To understand the role of ubiquitination in budding, we mutated the four lysines in MA to arginines and identified lysine 74 as the unique site of ubiquitination. Virus-like particles produced by the K74R mutant did not contain ubiquitinated MA and showed a fourfold reduction in the release of infectious particles. Furthermore, the K74R mutation rendered assembly hypersensitive to C2WW inhibition; K74R Gag budding was inhibited at significantly lower levels of expression of C2WW compared with wild-type Gag. This finding indicates that the interaction between Gag and WWP1 is required for functions other than Gag ubiquitination. Additionally, we show that the PPPY(-) mutant Gag exerts a strong dominant-negative effect on the budding of wild-type Gag, further supporting the importance of recruitment of WWP1 to achieve particle assembly.

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).

Three late assembly domain consensus motifs, namely PTAP, PPPY, and LYPXL, have been identified in different retroviruses. They have been shown to interact with the cellular proteins TSG101, Nedd4, and AP2 or AIP, respectively. Human T-cell leukemia virus type 1 (HTLV-1) has a PPPY and a PTAP motif, separated by two amino acids, located at the end of MA, but only the PPPY motif is conserved in the deltaretrovirus group. Like other retroviral peptides carrying the late motif, MA is mono- or di-ubiquitinated. A mutational analysis showed that 90% of PPPY mutant particles were retained in the cell compared to 15% for the wild-type virus. Mutations of the PTAP motif resulted in a 20% decrease in particle release. In single-cycle infectivity assays, the infectious titers of late motif mutants correlated with the amounts of released virus, as determined by an enzyme-linked immunosorbent assay. We observed binding of MA to the WW domains of the Nedd4 family member WWP1 but not to the amino-terminal ubiquitin E2 variant domain of TSG101 in mammalian two-hybrid analyses. The binding to WWP1 was eliminated when the PPPY motif was mutated. However, MA showed binding to TSG101 in the yeast two-hybrid system that was dependent on an intact PTAP motif. A dominant-negative (DN) mutant of WWP1 could inhibit budding of the intact HTLV-1 virus. In contrast, DN TSG101 only affected the release of virus-like particles encoded by Gag expression plasmids. Electron and fluorescent microscopy showed that Gag accumulates in large patches in the membranes of cells expressing viruses with PPPY mutations. Very few tethered immature particles could be detected in these samples, suggesting that budding is impaired at an earlier step than in other retroviruses.

A single-cycle infection assay with recombinant viral vectors was developed to study human T cell leukemia virus type I (HTLV-I) replication and its inhibition by antiviral agents. The susceptibility of HTLV-I to 6 nucleoside reverse-transcriptase inhibitors was examined. HTLV-I replication was inhibited by tenofovir, abacavir, lamivudine, zalcitabine, stavudine, and zidovudine.

The genomic RNA of human T-cell leukemia virus type 1 encodes three polyproteins, Gag, Gag-Pro, and Gag-Pro-Pol, which are translated as a result of no, one, and two frameshifts, respectively. In this report we demonstrate that the 77 residues encoded at the C terminus of the Gag-Pro precursor can be collectively detected as an 8-kDa transframe protein (TFP) in virions. Mutant viruses with a C-terminally truncated TFP (19, 32, or 50 residues) had essentially a wild-type phenotype in vitro. However, a virus mutant that encoded only the Gag and Gag-Pro-Pol polyproteins due to a mutation in the second frameshift site, and hence did not produce TFP, was noninfectious. Mutation analysis of the proteolytic cleavage site between PR and TFP revealed the presence of an additional site and the existence of a p1 peptide separating protease and TFP. While removal of the cleavage site at the PR-p1 junction had a modest effect on virus replication, mutation of the p1-TFP cleavage site led to noninfectious virus and the loss of reverse transcriptase activity. Determination of the amino-terminal sequence of a hemagglutinin-tagged RT demonstrated that the same site is used in processing the Gag-Pro-Pol precursor and thus defines the start of mature RT. Neither mutation alone or in combination caused changes in the amounts or processing patterns of the Gag polyprotein, indicating that protease is active independent of its C terminus.

A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37 degrees C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.

We have compared nef gene sequences isolated by PCR from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically or molecularly cloned SIV(Mne). Two samples from each animal obtained either early after infection (week 2-8) or after significant CD4+ depletion (week 21-137) were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two more in all but one. Two of the common exchanges are located about 40 residues apart in the Nef core sequence, but are in proximity on the tertiary structure as judged by computer modelling using the structure of the HIV Nef core protein as a guide. Most recurring in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIVsm/HIV-2 groups. Animals inoculated with virus already containing the "late version" nef gene developed a more aggressive disease. The macaque adapted (MA)nef conferred a threefold higher infectivity to the cloned virus, but had no effects on CD4 downregulation. Propagation of virus with MAnef in tissue culture resulted in the rapid emergence of variants with newly attenuated nef. These findings suggest that the selective pressure on nef in vivo and in vitro are different.

The protein kinase cascade Raf-MAPKK/MEK-MAPK/ERK connects protein tyrosine kinase receptors in the membrane with control of transcription factor activity in the nucleus. We have examined whether Raf is obligatory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope-tagged ERK-1 cDNA and a dominant inhibitory mutant of Raf-1 we found that serum and 12-O-tetradecanoylphorbol-13-acetate as well as representatives of three classes of oncogenes (protein tyrosine kinases abl/src, Ras, and protein serine/threonine kinases mos/cot) were all Raf-dependent for stimulation of MAPK. All of the MAPK stimulating oncogenes were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPK/ERK. Furthermore, the ability to stimulate MAPK/ERK appears to be critical for transformation by oncogenic Raf-1 and ERK-1 and -2 synergized with v-raf in a focus induction assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibitory. Raf/ERK synergism was also observed in transcriptional transactivation of the oncogene-response element in the polyoma enhancer. We conclude that this Raf signaling pathway, which connects to many upstream activators and downstream effectors, is essential for transformation by most oncogenes.