Evidence for Decreased Nucleolar PARP-1 as an Early Marker of Cognitive Impairment.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that regulates gene expression through poly(ADP)-ribosylation, resulting in the loosening of chromatin structure. PARP-1 enzymatic activity has been shown to be necessary for the expression of several genes required for memory formation and consolidation. Previously, we showed that nucleolar PARP-1 is significantly decreased in hippocampal pyramidal cells in Alzheimer's disease (AD). We proposed that the displacement of PARP-1 from the nucleolus results in downregulation of new rRNA expression and ribosome biogenesis, leading to cognitive impairment. To further investigate the relationship between nucleolar PARP-1 and memory impairment, we examined PARP-1 expression in the hippocampi of individuals with mild cognitive impairment (MCI) compared to control and AD cases. We used immunohistochemical techniques to examine the nucleolar distribution of PARP-1 in the Cornu Ammonis (CA region) of the hippocampus. PARP-1 positive cells were then scored for the presence or absence of PARP-1 in the nucleolus. We found a significant decrease of PARP-1 staining in the nucleolar compartment of hippocampal pyramidal cells in MCI compared with Control and AD. When the four CA (CA1-4) regions were considered separately, only the CA1 region showed significant differences in nucleolar PARP-1 with Control > AD > MCI cases. Categorization of nucleolar PARP-1 into "distinct" and "diffuse" groups suggest that most of the changes occur within the distinct group. In addition, measurements of the nucleolar diameter of nucleolar PARP-1 positive cells in CA2 and CA4 showed Control > MCI. Thus, MCI cases had a lower percentage of PARP-1 nucleolar positive cells in CA1 and smaller nucleolar diameters in CA2 and CA4, compared to Control. Our data suggest that disruption of nucleolar form and function is an early and important step in the progression of cognitive impairment.

Liver serine palmitoyltransferase activity deficiency in early life impairs adherens junctions and promotes tumorigenesis.

Serine palmitoyltransferase is the key enzyme in sphingolipid biosynthesis. Mice lacking serine palmitoyltransferase are embryonic lethal. We prepared liver-specific mice deficient in the serine palmitoyltransferase long chain base subunit 2 gene using an albumin-cyclization recombination approach and found that the deficient mice have severe jaundice. Moreover, the deficiency impairs hepatocyte polarity, attenuates liver regeneration after hepatectomy, and promotes tumorigenesis. Importantly, we show that the deficiency significantly reduces sphingomyelin but not other sphingolipids in hepatocyte plasma membrane; greatly reduces cadherin, the major protein in adherens junctions, on the membrane; and greatly induces cadherin phosphorylation, an indication of its degradation. The deficiency affects cellular distribution of ß-catenin, the central component of the canonical Wnt pathway. Furthermore, such a defect can be partially corrected by sphingomyelin supplementation in vivo and in vitro. CONCLUSION: The plasma membrane sphingomyelin level is one of the key factors in regulating hepatocyte polarity and tumorigenesis. (Hepatology 2016;64:2089-2102).

Nucleolar PARP-1 Expression Is Decreased in Alzheimer's Disease: Consequences for Epigenetic Regulation of rDNA and Cognition.

Synaptic dysfunction is thought to play a major role in memory impairment in Alzheimer's disease (AD). PARP-1 has been identified as an epigenetic regulator of plasticity and memory. Thus, we hypothesize that PARP-1 may be altered in postmortem hippocampus of individuals with AD compared to age-matched controls without neurologic disease. We found a reduced level of PARP-1 nucleolar immunohistochemical staining in hippocampal pyramidal cells in AD. Nucleolar PARP-1 staining ranged from dispersed and less intense to entirely absent in AD compared to the distinct nucleolar localization in hippocampal pyramidal neurons in controls. In cases of AD, the percentage of hippocampal pyramidal cells with nucleoli that were positive for both PARP-1 and the nucleolar marker fibrillarin was significantly lower than in controls. PARP-1 nucleolar expression emerges as a sensitive marker of functional changes in AD and suggests a novel role for PARP-1 dysregulation in AD pathology.

Ectopic pituitary adenoma associated with an empty sella presenting with hearing loss: case report with literature review.

Ectopic pituitary adenomas are uncommon entities that may pose substantial diagnostic challenges. In the majority of these cases, patients present with endocrine and/or nasal obstruction symptoms. We report the case of an ectopic pituitary adenoma in a 76-year-old man with an empty sella who initially presented with right-sided hearing loss progressing to bilateral hearing loss over the next 4 years. Neuroimaging studies revealed a large, expansile central skull base mass replacing the clivus and sphenoid sinus, and invading the internal auditory canals and inner ear bilaterally. The tumor also involved the floor of the middle cranial fossae and bilateral medial temporal and occipital bones. Histopathologic examination, including immunohistochemical studies, revealed a sparsely granulated lactotroph adenoma. Hearing loss in a patient with ectopic pituitary adenoma constitutes an extremely unusual presentation. This case was further complicated by the presence of an empty sella and the absence of symptoms related to hyperprolactinemia.

Correlation of Automated Chemiluminescent Method with Enzyme-Linked Immunosorbent Assay (ELISA) Antibody Titers in Convalescent COVID-19 Plasma Samples: Development of Rapid, Cost-Effective Semi-Quantitative Diagnostic Methods.

BACKGROUND: We investigated the utility of an automated chemiluminescent SARS-CoV-2 IgG antibody assay platform in quantifying the amount of binding antibodies present in donated convalescent plasma. METHODS: A total of 179 convalescent plasma units were analyzed for the presence of SARS-CoV-2 IgG antibodies using the Beckman-Coulter chemiluminescent immunoassay (CLIA) platform. The equipment-derived numerical values (S/Co ratio) were recorded. Aliquots from the same units were subjected to enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 S1 protein. The relationship between ELISA titers and CLIA S/Co values was analyzed using linear regression and receiver operating characteristics (ROC) curve. RESULTS: Twenty-one samples (11.7%) had S/Co values of less than 1.0 and were deemed negative for antibodies and convalescent plasma had S/Co values between >1.0 and 5.0 (70/179, 39.1%). Fifteen units (8.4%) had negative ELISA titer. The majority of the units (95/179. 53.1%) had titers ≥1:1024. The sensitivities of ELISA to CLIA were comparable (90.5% vs 88.3%, respectively; p=0.18). There was positive linear correlation between CLIA S/Co values and ELISA IgG titer (Rho = 0.75; Spearman's rank = 0.82, p-value = <0.0001). The agreement between the two methods was fair, with a κ index of 0.2741. Using the ROC analysis, we identified a CLIA S/Co cutoff value of 8.2, which gives a sensitivity of 90% and a specificity of 82% in predicting a titer dilution of ≥1:1024. CONCLUSION: The utility of automated antibody detection systems can be extended from simply a screening method to a semi-quantitative and quantitative functional antibody analysis. CLIA S/Co values can be used to reliably estimate the ELISA antibody titer. Incorporation of chemiluminescent-based methods can provide rapid, cost-effective means of identifying anti-SARS-CoV-2 antibody titers in donated plasma for use in the treatment of COVID-19 infection.

Administration of high titer convalescent anti-SARS-CoV-2 plasma: From donor selection to monitoring recipient outcomes.

Early in the SARS-CoV-2 pandemic, convalescent plasma (CP) therapy was proposed as a treatment for severely ill patients. We conducted a CP treatment protocol under the Mayo Clinic Extended Access Program at University Hospital Brooklyn (UHB). Potential donors were screened with a lateral flow assay (LFA) for IgM and IgG antibodies against the SARS-CoV-2 S1 receptor-binding domain (RBD). Volunteers that were LFA positive were tested with an ELISA to measure IgG titers against the RBD. Subjects with titers of at least 1:1024 were selected to donate. Most donors with positive LFA had acceptable titers and were eligible to donate. Out of 171 volunteers, only 65 tested positive in the LFA (38.0%), and 55 (32.2%) had titers of at least 1:1024. Before our donation program started, 31 CP units were procured from the New York Blood Center (NYBC). Among the 31 CP units that were obtained from the NYBC, 25 units (80.6%) were positive in the LFA but only 12 units (38.7%) had titers of at least 1:1024. CP was administered to 28 hospitalized COVID-19 patients. Patients who received low titer CP, high titer CP and patients who did not receive CP were followed for 45 days after presentation. Severe adverse events were not associated with CP transfusion. Death was a less frequent outcome for patients that received high titer CP (>1:1024) 38.6% mortality, than patients that received low titer CP (≤1:1024) 77.8% mortality.

The New York State SARS-CoV-2 Testing Consortium: Regional Communication in Response to the COVID-19 Pandemic.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, created an unprecedented need for comprehensive laboratory testing of populations, in order to meet the needs of medical practice and to guide the management and functioning of our society. With the greater New York metropolitan area as an epicenter of this pandemic beginning in March 2020, a consortium of laboratory leaders from the assembled New York academic medical institutions was formed to help identify and solve the challenges of deploying testing. This report brings forward the experience of this consortium, based on the real-world challenges which we encountered in testing patients and in supporting the recovery effort to reestablish the health care workplace. In coordination with the Greater New York Hospital Association and with the public health laboratory of New York State, this consortium communicated with state leadership to help inform public decision-making addressing the crisis. Through the length of the pandemic, the consortium has been a critical mechanism for sharing experience and best practices in dealing with issues including the following: instrument platforms, sample sources, test performance, pre- and post-analytical issues, supply chain, institutional testing capacity, pooled testing, biospecimen science, and research. The consortium also has been a mechanism for staying abreast of state and municipal policies and initiatives, and their impact on institutional and laboratory operations. The experience of this consortium may be of value to current and future laboratory professionals and policy-makers alike, in dealing with major events that impact regional laboratory services.

Implementation of a pooled surveillance testing program for asymptomatic SARS-CoV-2 infections in K-12 schools and universities.

BACKGROUND: The negative impact of continued school closures during the height of the COVID-19 pandemic warrants the establishment of cost-effective strategies for surveillance and screening to safely reopen and monitor for potential in-school transmission. Here, we present a novel approach to increase the availability of repetitive and routine COVID-19 testing that may ultimately reduce the overall viral burden in the community. METHODS: We implemented a testing program using the SalivaClear࣪ pooled surveillance method that included students, faculty and staff from K-12 schools (student age range 5-18 years) and universities (student age range >18 years) across the country (Mirimus Clinical Labs, Brooklyn, NY). The data analysis was performed using descriptive statistics, kappa agreement, and outlier detection analysis. FINDINGS: From August 27, 2020 until January 13, 2021, 253,406 saliva specimens were self-collected from students, faculty and staff from 93 K-12 schools and 18 universities. Pool sizes of up to 24 samples were tested over a 20-week period. Pooled testing did not significantly alter the sensitivity of the molecular assay in terms of both qualitative (100% detection rate on both pooled and individual samples) and quantitative (comparable cycle threshold (Ct) values between pooled and individual samples) measures. The detection of SARS-CoV-2 in saliva was comparable to the nasopharyngeal swab. Pooling samples substantially reduced the costs associated with PCR testing and allowed schools to rapidly assess transmission and adjust prevention protocols as necessary. In one instance, in-school transmission of the virus was determined within the main office and led to review and revision of heating, ventilating and air-conditioning systems. INTERPRETATION: By establishing low-cost, weekly testing of students and faculty, pooled saliva analysis for the presence of SARS-CoV-2 enabled schools to determine whether transmission had occurred, make data-driven decisions, and adjust safety protocols. We provide strong evidence that pooled testing may be a fundamental component to the reopening of schools by minimizing the risk of in-school transmission among students and faculty. FUNDING: Skoll Foundation generously provided funding to Mobilizing Foundation and Mirimus for these studies.

Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.

Retinol-binding protein 2 (RBP2) binds monoacylglycerols and modulates gut endocrine signaling and body weight.

Expressed in the small intestine, retinol-binding protein 2 (RBP2) facilitates dietary retinoid absorption. Rbp2-deficient (Rbp2-/- ) mice fed a chow diet exhibit by 6-7 months-of-age higher body weights, impaired glucose metabolism, and greater hepatic triglyceride levels compared to controls. These phenotypes are also observed when young Rbp2-/- mice are fed a high fat diet. Retinoids do not account for the phenotypes. Rather, RBP2 is a previously unidentified monoacylglycerol (MAG)-binding protein, interacting with the endocannabinoid 2-arachidonoylglycerol (2-AG) and other MAGs with affinities comparable to retinol. X-ray crystallographic studies show that MAGs bind in the retinol binding pocket. When challenged with an oil gavage, Rbp2-/- mice show elevated mucosal levels of 2-MAGs. This is accompanied by significantly elevated blood levels of the gut hormone GIP (glucose-dependent insulinotropic polypeptide). Thus, RBP2, in addition to facilitating dietary retinoid absorption, modulates MAG metabolism and likely signaling, playing a heretofore unknown role in systemic energy balance.

Serving as a Temporary Pathology Chair: "Boon" or "Boondoggle"?

The 2019 Association of Pathology Chairs Annual Meeting included a discussion group sponsored by the Senior Fellows Group (former chairs of academic departments of pathology who have remained active in Association of Pathology Chairs) that was focused on serving as temporary pathology chair. Such positions include "acting chair" (service while the permanent chair is on leave or temporarily indisposed), "interim chair" (service after departure of the prior chair and before a new chair is appointed), "term-limited chair" (usually one nonrenewable term of less than 5 years), and "terminal chair" (permanent chair being asked to stay until a successor is appointed). Discussion group panelists represented each of these positions and included the perspective of 3 former deans about the rationale for making such appointments. The potential benefits and risks of serving in these roles were discussed. Issues addressed included acting as "caretaker manager" or "change-agent leader"; whether such service and experience would enhance or harm one's chances to become a permanent chair of that or another department; the effect of such service on academic productivity; the influence of department and institutional factors on the position; the range of authority provided, particularly in addressing significant problems affecting the department's future; and the impact of time served in these various positions. The "lame-duck" effect of prolonged service as "terminal chair" was also discussed. The observations and advice provided by the panelists and audience discussion are reported and may be useful for those considering service as temporary chair in pathology as well as other academic leadership positions.

The Long-Term Persistence of Borrelia burgdorferi Antigens and DNA in the Tissues of a Patient with Lyme Disease.

Whether Borrelia burgdorferi, the causative agent of Lyme disease, can persist for long periods in the human body has been a controversial question. The objective of this study was to see if we could find B. burgdorferi in a Lyme disease patient after a long clinical course and after long-term antibiotic treatment. Therefore, we investigated the potential presence of B. burgdorferi antigens and DNA in human autopsy tissues from a well-documented serum-, PCR-, and culture-positive Lyme disease patient, a 53-year-old female from northern Westchester County in the lower Hudson Valley Region of New York State, who had received extensive antibiotic treatments during extensive antibiotic treatments over the course of her 16-year-long illness. We also asked what form the organism might take, with special interest in the recently found antibiotic-resistant aggregate form, biofilm. We also examined the host tissues for the presence of inflammatory markers such as CD3+ T lymphocytes. Autopsy tissue sections of the brain, heart, kidney, and liver were analyzed by histological and immunohistochemical methods (IHC), confocal microscopy, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), and whole-genome sequencing (WGS)/metagenomics. We found significant pathological changes, including borrelial spirochetal clusters, in all of the organs using IHC combined with confocal microscopy. The aggregates contained a well-established biofilm marker, alginate, on their surfaces, suggesting they are true biofilm. We found B. burgdorferi DNA by FISH, polymerase chain reaction (PCR), and an independent verification by WGS/metagenomics, which resulted in the detection of B. burgdorferi sensu stricto specific DNA sequences. IHC analyses showed significant numbers of infiltrating CD3+ T lymphocytes present next to B. burgdorferi biofilms. In summary, we provide several lines of evidence that suggest that B. burgdorferi can persist in the human body, not only in the spirochetal but also in the antibiotic-resistant biofilm form, even after long-term antibiotic treatment. The presence of infiltrating lymphocytes in the vicinity of B. burgdorferi biofilms suggests that the organism in biofilm form might trigger chronic inflammation.

Novel Selective Calpain 1 Inhibitors as Potential Therapeutics in Alzheimer's Disease.

Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-ß, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-ß elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.

Magnesium blocks the loss of protein kinase C, leads to a transient translocation of PKC(alpha) and PKC(epsilon), and improves recovery after anoxia in rat hippocampal slices.

Magnesium is a potent neuroprotective agent against damage to synaptic transmission during cerebral anoxia and reoxygenation. We investigated the mechanisms of anoxic transmission damage and magnesium neuroprotection by examining the response of PKC isoforms to an anoxic insult in the rat hippocampal slice model. A 2-min anoxic period, which resulted in almost complete recovery of synaptic function, did not result in PKC downregulation. In contrast, inducing long-term damage with 10-min anoxia resulted in the downregulation of the conventional PKCs betaI, betaII and gamma immediately after the insult and after 1-h reoxygenation. There was additional loss of PKC(alpha) and PKC(epsilon) after 1-h reoxygenation. Magnesium treatment improved the recovery of synaptic transmission, blocked the loss of PKC and resulted in a transient translocation of PKC(alpha) and PKC(epsilon) to the membrane fraction. Selective downregulation of cPKCs and PKC(epsilon) correlated with permanent damage to synaptic transmission while translocation of PKC(alpha) and PKC(epsilon) correlated with preservation of synaptic function. The mechanisms of magnesium neuroprotection may include altering the PKC response to an anoxic insult.

Endovascular external carotid artery occlusion for brain selective targeting: a cerebrovascular swine model.

BACKGROUND: The choice of an animal model for cerebrovascular research is often determined by the disease subtype to be studied (e.g. ischemic stroke, hemorrhage, trauma), as well as the nature of the intervention to be tested (i.e. medical device or pharmaceutical). Many initial studies are performed in smaller animals, as they are cost-effective and their encephalic vasculature closely models that of humans. Non-human primates are also utilized when confirmation or validation is required on higher levels and to test larger devices. However, working with primates is complex and expensive. Intermediate sized animal models, such as swine and sheep, may represent a valuable compromise. Their cerebrovascular anatomy, however, comes with challenges because of the natural higher external carotid artery perfusion and the existence of a rete mirabile. We describe a modification to the traditional swine cerebrovascular model that significantly enhances selective brain hemispheric perfusion, limiting external carotid perfusion and dilution. RESULTS: We investigated whether unilateral endovascular coil-embolization of external carotid artery branches in swine would lead to increased brain perfusion, altering cerebral circulation so that it more closely models human cerebral circulation. Equal amounts of approximately 4 °C cold saline were injected in 6 Yorkshire pigs into the ipsilateral common carotid artery before and after embolization. Hemispheric temperature changes from pre- and post-embolization were obtained as a measure of brain perfusion and averaged and compared using non-parametric statistical tests (Wilcoxon signed rank test, Mann-Whitney U Test). Graphs were plotted with absolute changes in hemispheric temperature over time to determine peak temperature drop (PTD) and corresponding time to peak (TTP) following the cold bolus injection. There was a 288 ± 90% increase in ipsilateral brain cooling after embolization indicating improved selective blood flow to the brain due to this vascular modification. CONCLUSION: We have developed an effective, selective vascular brain model in swine that may be useful as a practical and cost-reducing intermediate step for evaluating target dose-responses for central nervous system drugs and brain selective interventions, such as local hypothermia.

Feasibility of near-infrared spectroscopic tomography for intraoperative functional cerebral monitoring: a primate study.

OBJECTIVE: The wide-ranging manipulations to the cardiovascular system that frequently occur during cardiac surgery can expose the brain to variations in its blood supply that could prove deleterious. As a first step to developing a resource suitable for monitoring such changes, we detected the hemodynamic events induced in the brain of a primate model, using high-density near-infrared spectroscopy combined with tomographic reconstruction methods and validated the findings using established radiologic and histologic techniques. METHODS: Continuous monitoring of the relative changes in the components of the cerebral hemoglobin signal was performed using high-density near-infrared spectroscopy (270 source-detector channel array) in anesthetized bonnet macaques with the brain exposed to induced ischemia and other acute events. A comparative analysis (exact binomial test) applied to reconstructed 3-dimensional images before and after the events and between cerebral hemispheres, combined with postprocedure magnetic resonance imaging, and postmortem histopathologic examination of the macaques' brains was performed to document and validate the spatial features revealed by the optical findings. RESULTS: Relative changes in the measured and calculated components of the hemoglobin signal, in response to the performed manipulations, revealed substantial concurrence among the reconstructed 3-dimensional images, magnetic resonance imaging of the macaques' brains, and postmortem histopathologic examination findings. Concurrence was seen when the manipulated hemoglobin concentration and associated oxygenation levels were either increased or decreased, and whether they were bilateral or restricted to a specified hemisphere. CONCLUSIONS: Continuous near-infrared spectroscopy tomography has been shown to accurately capture and localize cerebral ischemia, vasodilatation, and hemorrhage in primates in real time. These findings are directly applicable to clinical intraoperative functional cerebral monitoring.

Multiple system atrophy in a patient with the spinocerebellar ataxia 3 gene mutation.

The cerebellar variant of multiple system atrophy (MSA-C) has overlapping clinical features with the hereditary spinocerebellar ataxias (SCAs), but can usually be distinguished on a clinical basis. We describe a patient who developed a sporadic, late-onset, rapidly progressive neurodegenerative disorder consistent with MSA-C. Genetic testing, however, showed an abnormal expansion of one allele of the spinocerebellar ataxia 3 (SCA3) gene. The clinical impression of MSA-C was confirmed by identification of numerous alpha-synuclein-containing glial cytoplasmic inclusions on autopsy. These findings suggest that abnormal expansion of the SCA3 gene may be a risk factor for the development of MSA-C.