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α-l-(3′-2′)-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitroprimer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3′ → 2′ versus 5′ → 3′, respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3′-OH of the primer’s terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primer’s terminal 3′-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

The increasing performance requirements of modern applications place a significant burden on software-based packet processing. Most of today’s software input/output accelerations achieve high performance at the expense of reserving CPU resources dedicated to continuously poll the Network Interface Card. This is specifically the case with DPDK (Data Plane Development Kit), probably the most widely used framework for software-based packet processing today. The approach presented in this paper, descriptively called Metronome, has the dual goals of providing CPU utilization proportional to the load, and allowing flexible sharing of CPU resources between I/O tasks and applications. Metronome replaces DPDK’s continuous polling with an intermittent sleep&wake mode, and revolves around a new multi-threaded operation, which improves service continuity. Since the proposed operation trades CPU usage with buffering delay, we propose an analytical model devised to dynamically adapt the sleep&wake parameters to the actual traffic load, meanwhile providing a target average latency. Our experimental results show a significant reduction of the CPU cycles, improvements in power usage, and robustness to CPU sharing even when challenged with CPU-intensive applications.

Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson’s disease (PD). Iron-loaded microglia are frequently found in affected brain regions, but how iron accumulation influences microglia physiology and contributes to neurodegeneration is poorly understood. Here we show that human induced pluripotent stem cell-derived microglia grown in a tri-culture system are highly responsive to iron and susceptible to ferroptosis, an iron-dependent form of cell death. Furthermore, iron overload causes a marked shift in the microglial transcriptional state that overlaps with a transcriptomic signature found in PD postmortem brain microglia. Our data also show that this microglial response contributes to neurodegeneration, as removal of microglia from the tri-culture system substantially delayed iron-induced neurotoxicity. To elucidate the mechanisms regulating iron response in microglia, we performed a genome-wide CRISPR screen and identified novel regulators of ferroptosis, including the vesicle trafficking gene SEC24B. These data suggest a critical role for microglia iron overload and ferroptosis in neurodegeneration.

Introduction: Healthcare expenditure is on the rise placing greater emphasis on operational excellence, cost containment, and high quality of care. Significant variation is seen in operating room (OR) costs with common surgical procedures such as laparoscopic appendectomy. Surgeons can influence cost through the selection of instrumentation for common surgical procedures such as laparoscopic appendectomy. We aimed to quantify the cost of laparoscopic appendectomy in our healthcare system and compare cost variations to operative times and outcomes. Methods and procedures: We performed a retrospective review of laparoscopic appendectomies in a large regional healthcare system during one-year period (2018). Operating room supply costs and procedure durations were obtained for each hospital. The American College of Surgeons National Surgical Quality Improvement Program (NSQIP) outcomes and demographics were compared to the costs for each hospital. Results: A total of 4757 laparoscopic appendectomies were performed at 20 hospitals (27 to 522 per hospital) by 233 surgeons. The average supply cost per case ranged from $650 to $1067. Individual surgeon cost ranged from $197 to $1181. The average operative time was 41 min (range 33 to 60 min). There was no association between lower cost and longer operative time. The patient demographics and comorbidities were similar between sites. There were no significant differences in postoperative complications between high- and low-cost centers. The items with the greatest increase in cost were single-use energy devices (SUD) and endoscopic stapler. We estimate that a saving of over $417 per case is possible by avoiding the use of energy devices and may be as high as $ 984 by adding selective use of staplers. These modifications would result in an annual savings of $1 million for our health system and more than $ 125 million nationwide. Conclusion: Performing laparoscopic appendectomy with reusable instruments and finding alternatives to expensive energy devices and staplers can significantly decrease costs and does not increase operative time or postoperative complications.

Gene therapy holds great promise for the treatment of acquired genetic disorders such as cancer with reduced side effects compared to chemotherapy. For gene therapy to be successful, it is crucial to develop efficient and nontoxic gene carriers to overcome the poor in vivostability and low cellular uptake of nucleic acid-based therapeutic agents. Here, we report a new and versatile approach exploring a combination of hydrophobic modifications and dual-stimuli-responsive degradation (SRD) for controlled gene delivery with amphiphilic block copolymer-based nanocarriers. The block copolymer, synthesized by atom transfer radical polymerization, is designed with an acid-labile acetal linkage at the block junction and a pendant disulfide group in the hydrophobic block. The incorporation of labile linkages enables both disulfide-core-cross-linking and dual-location dual-acid/reduction-responsive degradation (DL-DSRD). Furthermore, the disulfide linkages integrated as hydrophobic moieties facilitate the nucleic acids to condense into nanometer-sized micelleplexes through electrostatic interactions of pendant dimethylamino groups with the anionic phosphate groups of the nucleic acids. Our preliminary results demonstrate that the DL-DSRD approach through hydrophobic modification is a robust platform in the development of gene delivery systems with enhanced colloidal stability, reduced cytotoxicity, and improved gene transfection efficiency.

Lung is the second most common locationof cystic echinococcosis (CE), after the liver. Diagnosis of lung CE is often incidental, and clinical manifestations depend on the location and size of the cyst, the most common being chest pain, shortness of breath, expectoration of fragments of endocyst, and haemoptysis. Surgery is the primary treatment, with a minor role for medical therapy. Delayed diagnosis and treatment may have important consequences. We present a case of lung CE in whichsurgical treatment was delayed due to the first wave of COVID-19. Since surgery could not be performed immediately, the patient was kept on albendazole and the cyst stage moved from CE1 to CE3a, to CE4, eventually requiring a more aggressive pericystectomy instead of the commonly performed endocystectomy. The clinical and imaging characteristics of a rare CE4 cyst of the lung are reported.

Global cerebral ischemia occurs when blood flow to the entire brain is transiently blocked, which results in delayed neurologic deficits. Here, we present a protocol for performing the four-vessel occlusion rat model to study the neurodegeneration and cognitive deficits associated with global ischemia. We describe steps for carrying out the vertebral and common carotid artery occlusion which enables sufficient blockage of cerebral blood flow. We then detail expected outcomes using histology assays and behavioral tests.

Laparoscopic cholecystectomy is the most common procedure performed by general surgeons in the United States, with approximately 600,000 procedures performed annually. As the cost of care rises, there is increasing emphasis on utilization and quality. Our objective was to evaluate the cost of laparoscopic cholecystectomy in our health system and to compare the operative times and outcomes at high- and low-cost centers. We evaluated all laparoscopic cholecystectomies performed in our system over a 1-year period. The operating room supply costs and procedure durations were obtained for each of the hospitals. The American College of Surgeons National Surgical Quality Improvement Program outcomes and demographics were compared to the costs for each hospital. During the study period, 7601 laparoscopic cholecystectomies were performed at 20 hospitals (170–759/hospital) by 227 surgeons. The average cost per case ranged from $296 at the lowest cost center to $658 at the highest cost center. The average operative time varied between sites from 46 to 95 min. There was no association between cost and operative time or case volume. There was a slight trend toward increased cost with higher number of emergency procedures, but this was not well correlated (R2= 0.03). The patient demographics and comorbidities were similar between sites. There were no significant differences in postoperative complications between high- and low-cost centers. The items with the greatest increase in cost were disposable trocars, disposable hook cautery, disposable endoscissors, and disposable clip appliers. We estimate that a savings of over $300/case is possible by using reusable instruments, which would result in an annual savings of $1.3 million for our health system, and $285 million nationwide. Performing laparoscopic cholecystectomy with reusable instruments can significantly decrease costs and does not increase operative time or postoperative complications.

Supporting programmable stateful packet forwarding functions in hardware requires a tight balance between functionality and performance. Current state-of-the-art solutions are based on a very conservative model that assumes worst-case workloads. This finally limits the programmability of the system, even if actual deployment conditions may be very different from the worst-case scenario.We use trace-based simulations to highlight the benefits of accounting for specific workload characteristics. Furthermore, we show that relatively simple additions to a switching chip design can take advantage of such characteristics. In particular, we argue that introducing stalls in the switching chip pipeline enables stateful functions to be executed in a larger but bounded time without harming the overall forwarding performance. Our results show that, in some cases, the stateful processing of a packet could use 30x the time budget provided by state of the art solutions.

The fast evolving nature of modern cyber threats and network monitoring needs calls for new, “software-defined”, approaches to simplify and quicken programming and deployment of online (stream-based) traffic analysis functions. StreaMon is a carefully designed data-plane abstraction devised to scalably decouple the “programming logic” of a traffic analysis application (tracked states, features, anomaly conditions, etc.) from elementary primitives (counting and metering, matching, events generation, etc), efficiently pre-implemented in the probes, and used as common instruction set for supporting the desired logic. Multi-stage multi-step real-time tracking and detection algorithms are supported via the ability to deploy custom states, relevant state transitions, and associated monitoring actions and triggering conditions. Such a separation entails platform-independent, portable, online traffic analysis tasks written in a high level language, without requiring developers to access the monitoring device internals and program their custom monitoring logic via low level compiled languages (e.g., C, assembly, VHDL). We validate our design by developing a prototype and a set of simple (but functionally demanding) use-case applications and by testing them over real traffic traces.

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth, autophagy, translation, and survival. Dysregulation of mTOR signaling is associated with cancer, diabetes, and autism. However, a role for mTOR signaling in neuronal death is not well delineated. Here we show that global ischemia triggers a transient increase in mTOR phosphorylation at S2448, whereas decreasing p-mTOR and functional activity in selectively vulnerable hippocampal CA1 neurons. The decrease in mTOR coincides with an increase in biochemical markers of autophagy, pS317-ULK-1, pS14-Beclin-1, and LC3-II, a decrease in the cargo adaptor p62, and an increase in autophagic flux, a functional readout of autophagy. This is significant in that autophagy, a catabolic process downstream of mTORC1, promotes the formation of autophagosomes that capture and target cytoplasmic components to lysosomes. Inhibitors of the lysosomal (but not proteasomal) pathway rescued the ischemia-induced decrease in mTOR, consistent with degradation of mTOR via the autophagy/lysosomal pathway. Administration of the mTORC1 inhibitor rapamycin or acute knockdown of mTOR promotes autophagy and attenuates ischemia-induced neuronal death, indicating an inverse causal relation between mTOR, autophagy, and neuronal death. Our findings identify a novel and previously unappreciated mechanism by which mTOR self-regulates its own levels in hippocampal neurons in a clinically relevant model of ischemic stroke.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and heterologous immunization approaches implemented worldwide for booster doses call for diversified vaccine portfolios. GRAd-COV2 is a gorilla adenovirus-based COVID-19 vaccine candidate encoding prefusion-stabilized spike. The safety and immunogenicity of GRAd-COV2 is evaluated in a dose- and regimen-finding phase 2 trial (ClinicalTrials.gov: NCT04791423) whereby 917 eligible participants are randomized to receive a single intramuscular GRAd-COV2 administration followed by placebo, or two vaccine injections, or two doses of placebo, spaced over 3 weeks. Here, we report that GRAd-COV2 is well tolerated and induces robust immune responses after a single immunization; a second administration increases binding and neutralizing antibody titers. Potent, variant of concern (VOC) cross-reactive spike-specific T cell response peaks after the first dose and is characterized by high frequencies of CD8s. T cells maintain immediate effector functions and high proliferative potential over time. Thus, GRAd vector is a valuable platform for genetic vaccine development, especially when robust CD8 response is needed.

By leveraging the uneven distribution of traffic among network flows, the authors improve the query throughput of Cuckoo hashing. They achieve this by placing the most frequently used items in the table that's accessed first during the Cuckoo query operation. Their scheme is named Cuckoo cache, as it's conceptually similar to a cache but implemented inside the Cuckoo hash with little additional cost. Cuckoo cache is evaluated using a traffic monitoring application fed with real traces. The results are compared with existing Cuckoo hashing implementations and show significant improvements.

The lack of Obsessive-Compulsive Disorder (OCD) symptom measures validated for use with older adults has hindered research and treatment development for the age group. We evaluated the Obsessive-Compulsive Inventory-Revised (OCI-R; Foa et al., 2002) with participants aged 65 and older (N=180) to determine if the measure was an effective tool for evaluating obsessional symptoms. Participants completed the OCI-R and a comprehensive assessment battery up to four times over approximately 18 months. Results supported the well-replicated latent structure of the OCI-R (i.e., Washing, Checking, Ordering, Obsessing, Hoarding, and Neutralizing). OCI-R total score was robustly associated with OCD symptoms assessed 18 months later by clinical interview, while scores on self-report measures of worry, general anxiety, and depression were not. Results indicate the OCI-R is an effective OCD symptom measure for older adults, although replication with additional older adult samples is needed.

The synthesis and incorporation of a tosylated phosphoramidite linker containing a disulfide bond is described. Incorporation of the linker into short DNA and RNA oligomers proceeded efficiently using automated solid phase synthesis. Treatment of the support bound oligonucleotide followed by cleavage from the solid support provided a variety of common functional handles, expanding the strategies of bifunctional modification of synthetic oligonucleotides for conjugation applications.

This article was updated to a correct typo in Figure 3.

Abstract This paper addresses the design of storage systems for operation under critical environmental conditions. For these applications, these systems should have low latency time in access, high performance in throughput and high storage capabilities; therefore, they must be assembled using highly reliable components, while allowing flexibility in design. Commercial Off The Shelf (COTS) components have often been used. A COTS-based architecture is analyzed in this paper; the proposed architecture uses design-level techniques (such as error detection/correction codes and scrubbing) to make commercially available Dynamic Random Access Memory (DRAM) chips tolerant to faults. This paper provides a complete and novel analysis of engineering alternatives which arise in the design of a highly reliable memory system based on Reed Solomon coding. A comparative analysis of methods for permanent fault detection is provided; moreover using a Markovian characterization, different functional arrangements (based on code and scrubbing frequency) are investigated and evaluated.

Tissue transglutaminase (tTGase) is a GTP-binding Ca2+-dependent enzyme which catalyses the post-translational modification via ϵ(γ-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme.

Quail embryo chondrocytes in culture display two morphological phenotypes: polygonal epithelial-like and floating cells. Both cell populations synthesize cartilage extracellular matrix proteins (type II collagen and specific proteoglycans), whereas type X collagen, which appears to be a marker of later stages of chondrocyte differentiation, is expressed only by the epithelial-like cells. Avian myelocytomatosis virus strain MC29 does not induce morphological transformation in quail embryo chondrocytes but stimulates these cells to proliferate with a progressively reduced doubling time. MC29-infected chondrocytes can be established in culture as a continuous cell line, whereas control (uninfected) cultures only survive a few months. Rapidly dividing MC29-infected chondrocytes still express type II collagen and cartilage proteoglycans but do not synthesize type X collagen.

Retinoic acid (RA) treatment of a suspension of quail chondrocytes inhibits the expression of cartilage collagens and induces cell adhesion along with fibronectin expression. We asked whether the RA-induced modulation of the chondrocyte phenotype was dependent on cell adhesion. Prevention of cell adhesion blocks cell growth and many of the effects associated with RA, such as collagen II inhibition, collagen I activation and fibronectin induction. The activity of the bone/tendon promoter of the α2(I) collagen gene was determined by measuring the transient expression of COL1A2-CAT, a chimaeric gene bearing 3500 bp from upstream of the transcription start site of the human α2(I) gene fused to the chloramphenicol acetyltransferase (CAT) gene. This promoter is activated only in permissive conditions for cell adhesion. The attachment activities of chondrocytes on protein substrates was studied by an in vitro cell adhesion assay. Untreated cells or cells maintained in suspension while undergoing RA treatment do not attach when replated on protein substrates. Chondrocytes treated with RA in permissive conditions for cell adhesion rapidly attach and spread instead on collagen-coated wells. Altogether the results suggest that cell adhesion plays a major role in RA-induced modulation of the chondrocyte phenotype.

Growth of quail chondrocytes in the presence of retinoic acid (RA) results in the suppression of the differentiated phenotype. RA-treated chondrocytes recover their differentiated phenotype if they are cultured for an additional 15 days in the absence of RA. A few days after removal from RA, treated chondrocytes acquire the polygonal morphology characteristic of chondrocytes growing as attached cells; they also gradually resume collagen II expression and synthesize cultures. The levels of collagen X mRNA decrease during the second week of culture in the absence of RA. Finally, at the end of 15 days, the absolute levels of collagen II and collagen X mRNAs are very similar in control and recovering chondrocytes.

alpha 2(I) collagen gene expression is induced in quail embryo chondrocytes pretreated with retinoic acid (RA). The initial appearance of alpha 2(I) mRNA occurs around day 3 of culture in RA-free medium and rapidly progresses over the next 4 days. In transient transfection assays, expression of COL1A2-CAT, a chimeric gene bearing 3500 bp upstream the bone/tendon transcription start site from the human alpha 2(I) gene fused to the CAT gene, is stimulated severalfold in RA-treated chondrocytes. In contrast, enzyme activity is very low in untreated chondrocytes, suggesting that the sequences required for RA-induced transcription of the alpha 2(I) gene are present in this plasmid. Analysis of alpha 2(I) promoter sequences performed with deletion mutants gives overlapping results in collagen type I-producing fibroblasts and chondrocytes withdrawn from RA treatment. These experiments suggest that RA-induced transcription of the alpha 2(I) collagen gene in chondrocytes is regulated by the binding of transcription factors to the same regulatory sequences that control transcription in fibroblasts.

We have isolated from a single plaque a mutant of Sindbis virus characterized by an E1 glycoprotein with higher electrophoretic mobility. This higher mobility is not attributable to a different extent of glycosylation of the protein nor to an altered proteolytic maturation pathway of the polypeptide precursor, but is the result of a deletion occurring during the replication of the viral RNA. The 26S RNA (the messenger for the Sindbis structural proteins) extracted from cells infected with the mutant is about 0.75 x 10(5) daltons smaller than the 26S RNA from the parental strain. As a consequence, in cells infected with the mutant, an E1 glycoprotein is synthesized with a polypeptide chain about 70 amino acids shorter. The biological relevance of this naturally occurring deletion of the viral genome is discussed.

Nonaccidental trauma (NAT) is most common and most lethal in infants. Falls are the most frequently given explanation for NAT, and head injuries can result from both mechanisms. We hypothesized that infant head injuries from NAT have a distinct injury profile compared to falls.

TWO FIBEROPTIC instruments to be used for direct visualization of the structures of the middle ear are described. Direct visualization is of considerable value in the diagnosis and presurgical evaluation of middle ear disease. Until now, visual evaluation of the middle ear has been possible only by surgical entrance or by peephole views through perforations in the tympanic membrane. Also, some indirect knowledge of the middle ear has been obtainable from the appearance of the tympanic membrane.The two instruments developed for visualization of the middle ear fulfill the following specifications: (a) the diameter of the probe is small enough to permit insertion through a perforation or myringotomy; (b) the source of light for illumination of the specimen area is located externally so as not to damage the inner ear; and (c) an image of the specimen formed at the distal end of the probe is transmitted from the middle

A new instrument and method for amnioscopy is described. Amnioscopy increases the precision and safety of prenatal transfusion and opens new avenues for fetal research and the photographic recording of prenatal events.

An infrared polariscope was constructed for photoelastic stress analysis of semiconductor materials transmitting in the 0.7 μ to 1.2 μ spectral region. The instrument which functions as either a plane or circular polariscope permits specimens up to 40-mm diam to be studied in collimated radiation. Glan–Thompson prisms are used for the polarizer and analyzer, and the associated quarter-wave plates are made of quartz. An infrared image-converter tube is used to observe the stress patterns produced by induced and frozenin stresses of specimens. A mechanical stage provides for rotary motion and lateral displacement of the specimen in two mutually perpendicular directions in a plane normal to the optical axis of the polariscope, and also allows the specimen to be stressed by a calibrated mechanical load. A material fringe value of 27 kg-cm^−1 fringe^−1 was determined for silicon and used to estimate residual stresses in a boule of silicon. Photographs of isoclinic and isochromatic fringe patterns at a wavelength of 1.1 μ are presented.

The fractional sensitivity and operational refractive index range of the rod photorefractometer have been extended by physical modifications of axially symmetric refracting elements and the mode of illumination. Optical systems using conical and cylindrical rod elements with flat or spherical entrance ends illuminated by an axial light cone or a hollow annular light cone have been studied. Expressions describing the sensitivity of these configurations are presented, and typical numerical cases are computed on an IBM 650 computer. Extension of range to specimen index close to 1.0 permits the photorefractometer to detect index changes of low refractive index media. Experimental verification of the theory is achieved.

A study of the extension of the rod and cone photorefractometer principle to the measurement of N and K, the real and imaginary parts of refractive index of absorbing fluids, is made. Theoretical analysis indicates high sensitivity in the low-absorption-coefficient region of the specimen when the illumination is incident at the interface at an angle close to the critical angle. Experiments are made on various rod configurations and results agree closely with theory.

Receptor interacting protein kinase 1 (RIPK1) mediates cell death and inflammatory signaling and is increased in multiple sclerosis (MS) brain samples. Here, we investigate the role of glial RIPK1 kinase activity in mediating MS pathogenesis. We demonstrate RIPK1 levels correlate with MS disease progression. We find microglia are susceptible to RIPK1-mediated cell death and identify an inflammatory gene signature that may contribute to the neuroinflammatory milieu in MS patients. We uncover a distinct role for RIPK1 in astrocytes in regulating inflammatory signaling in the absence of cell death and confirm RIPK1-kinase-dependent regulation in human glia. Using a murine MS model, we show RIPK1 inhibition attenuates disease progression and suppresses deleterious signaling in astrocytes and microglia. Our results suggest RIPK1 kinase activation in microglia and astrocytes induces a detrimental neuroinflammatory program that contributes to the neurodegenerative environment in progressive MS.