Structural stability of SARS-CoV-2 virus like particles degrades with temperature.

SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter.

Structural stability of SARS-CoV-2 degrades with temperature.

SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter. STATEMENT OF SCIENTIFIC SIGNIFICANCE: The economic and public health impact of the COVID-19 pandemic are very significant. However scientific information needed to underpin policy decisions are limited partly due to novelty of the SARS-CoV-2 pathogen. There is therefore an urgent need for mechanistic studies of both COVID-19 disease and the SARS-CoV-2 virus. We show that individual virus particles suffer structural destabilization at relatively mild but elevated temperatures. Our nanoscale results are consistent with recent observations at larger scales. Our work strengthens the case for COVID-19 resurgence in winter.

Temperature-dependent activity of kinesins is regulable.

Cytoskeletal transport in cells is driven by enzymes whose activity shows sensitive, typically Arrhenius, dependence on temperature. Often, the duration and outcome of cargo transport is determined by the relative success of kinesin vs. dynein motors, which can simultaneously bind to individual cargos and move in opposite direction on microtubules. The question of how kinesin and dynein activity remain coupled over the large temperature ranges experienced by some cells is one of clear biological relevance. We report a break in the Arrhenius behavior of both kinesin-1 and kinesin-3 enzymatic activity at 4.7 °C and 10.5 °C, respectively. Further, we report that this transition temperature significantly changes as a function of chemical background: addition of 200 mM TMAO increases transition temperatures by ∼6 °C in all cases. Our results show that Arrhenius trend breaks are common to all cytoskeletal motors and open a broad question of how such activity transitions are regulated in vivo. STATEMENT OF SIGNIFICANCE: Many cytoskeletal motors studied to date follow Arrhenius kinetics, at least from room temperature up to mammalian body temperature. However the thermal dynamic range is typically finite, and breaks in Arrhenius trends are commonly observed at biologically relevant temperatures. Here we report that the thermal dynamic range of kinesins is also limited and moreover that the location of the Arrhenius break for kinesins can shift significantly based on chemical backgrounds. This implies that the balance of multiple motor cargo transport along the cytoskeleton is far more tunable as a function of temperature than previously appreciated.

Mutations in the tail domain of DYNC1H1 cause dominant spinal muscular atrophy.

OBJECTIVE: To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600). METHODS: Target exon capture and next generation sequencing was used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed. RESULTS: Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state-a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function. CONCLUSIONS: We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.

Quantitative NAT for pathogen inactivation verification.

A novel Quantitative Nucleic Acid Test (Q-NAT) technology has been developed to demonstrate, quantify and verify pathogen inactivation by methods that break pathogen nucleic acids, specifically, gamma irradiation. The Q-NAT technology provides significant advantages in cost, efficiency and broad applicability compared with traditional methods for pathogen inactivation detection and quantification such as cell culture.

Evaluating blunt abdominal trauma with sonography: a cost analysis.

Ultrasonography (US) is becoming increasingly utilized in the United States for the evaluation of blunt abdominal trauma (BAT). The objective of this study was to assess the cost impact of utilizing US in the evaluation of patients with BAT in a major trauma center. All patients sustaining BAT during a 6-month period before US was used at our institution (Jan-Jun 1993) were compared to BAT patients from a recent period in which US has been utilized (Jan-Jun 1995). The numbers of US, computed tomography (CT), and diagnostic peritoneal lavage (DPL) were tabulated for each group. Financial cost for each of these procedures as determined by our finance department were as follows: US $96, CT $494, DPL $137. These numbers are representative of actual hospital expenditures exclusive of physician fees as calculated in 1994 U.S. dollars. Cost analysis was performed with t test and chi squared test, and significance was defined as P < 0.05. There were 890 BAT admissions in the 1993 study period and 1033 admissions in the 1995 study period. During the 1993 period, 642 procedures were performed on the 890 patients to evaluate the abdomen: 0 US, 466 CT, and 176 DPL (see table) [table: see text]. This compares to 801 procedures on the 1,033 patients in 1995: 552 US, 228 CT, and 21 DPL. Total cost was $254,316 for the 1993 group and $168,501 for the 1995 group. Extrapolated to a 1-year period, a significant (P < 0.05) cost savings of $171,630 would be realized. Cost per patient evaluated was significantly reduced from $285.75 in 1993 to $163.12 in 1995 (P < 0.05). This represents a 43 per cent reduction in per patient expenditure for evaluating the abdomen. By effectively utilizing ultrasonography in the evaluation of patients with blunt abdominal trauma, a significant cost savings can be realized. This effect results chiefly from an eight-fold reduction in the use of DPL, and a two-fold reduction in the use of CT.

Hemoperitoneum score helps determine need for therapeutic laparotomy.

PURPOSE: Sonography provides a fast, portable, and noninvasive method for patient assessment. However, the benefit of providing real-time ultrasound (US) imaging and fluid quantification shortly after patient arrival has not been explored. The objective of this study was to prospectively validate a US hemoperitoneum scoring system developed at our institution and determine whether sonography can predict a therapeutic operation. METHODS: For 12 months, prospective data on all patients undergoing a trauma sonogram were recorded. All sonograms positive for free fluid were given a hemoperitoneum score. The US score was compared with initial systolic blood pressure and base deficit to assess the ability of sonography to predict a therapeutic laparotomy. RESULTS: Forty of 46 patients (87%) with a US score > or = 3 required a therapeutic laparotomy. Forty-six of 54 patients with a US score < 3 (85%) did not need operative intervention. The sensitivity of sonography was 83% compared with 28% and 49% for systolic blood pressure and base deficit, respectively, in determining the need for therapeutic operation. CONCLUSION: We conclude that the majority of patients with a score > or = 3 will need surgery. The US hemoperitoneum scoring system was a better predictor of a therapeutic laparotomy than initial blood pressure and/or base deficit.

2,576 ultrasounds for blunt abdominal trauma.

BACKGROUND: Determination of intra-abdominal injury following blunt abdominal trauma (BAT) continues to be a diagnostic challenge. Ultrasound (US) has been described as a potentially useful diagnostic tool in this setting and is being used with increasing frequency in trauma centers. We determined the diagnostic capability of US in the evaluation of BAT. METHODS: A retrospective analysis of our trauma US database was performed over a 30-month period. Computed tomographic scan, diagnostic peritoneal lavage, or exploratory laparotomy confirmed the presence of intra-abdominal injury. RESULTS: During the study period, 8,197 patients were evaluated at the Ryder Trauma Center. Of this group, 2,576 (31%) had US in the evaluation of BAT. Three hundred eleven (12%) US exams were considered positive. Forty-three patients (1.7%) had a false-negative US; of this group, 10 (33%) required exploratory laparotomy. US had a sensitivity of 86%, a specificity of 98%, and an accuracy of 97% for detection of intra-abdominal injuries. Positive predictive value was 87% and negative predictive value was 98%. CONCLUSION: Emergency US is highly reliable and may replace computed tomographic scan and diagnostic peritoneal lavage as the initial diagnostic modality in the evaluation of most patients with BAT.

Ultrasound of blunt abdominal trauma.

Widely accepted in Europe and Asia as a screening tool for blunt abdominal trauma, sonography is gradually gaining popularity among trauma and emergency physicians in the United States. Sonography has been shown to be comparable with DPL and CT for the detection of hemoperitoneum and superior to both modalities because of its rapidity, noninvasiveness, portability, and low cost. With its ability to demonstrate the amount of intraperitoneal hemorrhage within minutes of a patient's arrival, sonography may be considered the screening modality of choice for blunt abdominal trauma.

Determination of the conformations of cAMP receptor protein and its T127L,S128A mutant with and without cAMP from small angle neutron scattering measurements.

Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.

Mesenteric panniculitis: case report and literature review.

Mesenteric panniculitis is an extremely rare inflammatory condition of the adipose tissue of unknown etiology in which the mesentery is replaced with fibrosis. Knowledge of this rare syndrome should prevent any unwarranted aggressive therapy and help to use the clinical, radiological, and surgical sources to obtain the diagnosis. This paper is a review of symptomatology, pathology, treatment, and outcome of this disorder. A case report is described that presented with obstruction of the sigmoid colon.

Sonography as the primary screening technique for blunt abdominal trauma: experience with 899 patients.

OBJECTIVE: The purpose of this study was to determine the accuracy of sonography when used routinely as the primary screening technique for blunt abdominal trauma. SUBJECTS AND METHODS: From December 1994 through December 1995, we used sonography as the primary screening technique for the initial assessment of blunt abdominal trauma. Sonography was performed by dedicated technologists and overseen and interpreted by radiology residents or attending physicians. A positive study was defined as evidence of free fluid or parenchymal injury. Sonographic findings were correlated with CT, surgery, or clinical follow-up. RESULTS: Of the 899 sonograms obtained for blunt abdominal trauma, the findings of 783 were negative, 101 were positive, and 15 were indeterminate. Of the 783 sonograms with negative findings, 768 (98%) were confirmed by CT, surgery, or clinical follow-up. Similarly, of the 101 sonograms with positive findings, 95 (94%) were confirmed. Interpretations resulted in 15 false-negative and five false-positive examinations. For all sonograms, we calculated a sensitivity of 86%, a specificity of 99%, and an accuracy of 98%. CONCLUSION: Sonography can be used effectively as the primary screening technique for blunt abdominal trauma.

Can surgeons evaluate emergency ultrasound scans for blunt abdominal trauma?

OBJECTIVE: To determine whether surgeons and residents with minimal training can evaluate accurately emergency ultrasound (US) examinations compared with radiologists for blunt abdominal trauma. METHODS: Over 7 months, we conducted a prospective study comparing the evaluation of emergency US for blunt abdominal trauma by surgeons and attending radiologists. US readings from the surgical team and the radiologists were correlated with outcome. RESULTS: One hundred-twelve patients were included in the study. Ninety-two patients had an US read as negative by the surgical and radiology services with no subsequent injuries identified. Eighteen patients had an US deemed positive by the surgical service and radiologists. Injuries were confirmed in this group by operation or computed tomography. One patient had an US deemed positive by the surgical team and subsequently negative by the radiologist. A diagnostic peritoneal lavage was performed which was negative. Another patient had an US interpreted as negative by the surgical evaluator and positive by the radiologist. Exploratory laparotomy was negative for intraabdominal hemorrhage or organ injury. Overall results reveal an accuracy on US reading of 99% for the surgical team and 99% for the attending radiologists. CONCLUSION: Surgeons and surgical residents at different levels of training can accurately interpret emergency ultrasound examinations for blunt trauma from the real-time images, at a level comparable to attending radiologists.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

The complete genome sequence of the gastric pathogen Helicobacter pylori.

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

A whole genome shotgun gene fusion method for isolation of translation initiation sites in Escherichia coli: identification of Haemophilus influenzae translation initiation sites in E. coli.

We have developed a new method for isolating translation initiation sites based on the expression of Haemophilus influenzae Rd gene fusions with the Escherichia coli galactokinase (galK) gene. We cloned random DNA fragments of H. influenzae Rd DNA into a plasmid vector containing the galK coding sequence from which the translation initiation site (the ribosome binding site and translation initiation codon) had been removed. A subset of the cloned DNA fragments contained translation initiation sites that, when fused to the galK gene, produced active galactokinase and complemented the host galK mutation. Molecules expressing galactokinase activity were isolated and characterized by DNA sequence analysis, and the sequences were aligned with the recently completed whole genomic sequence of H. influenzae Rd. Translation initiation sites for known, hypothetical, and new genes were identified. Translation initiation sites internal to the coding sequences of a number of genes were identified, suggesting that internal translation initiation sites are common, especially in large genes. This shotgun method provides functional information on translation initiation sites and helps to define gene coding sequences.

Glutamate as a putative neurotransmitter in the mollusc, Lymnaea stagnalis.

Bath-applied glutamate (10-1000 microM) produced excitatory and inhibitory responses on numerous identified neurons of the mollusc Lymnaea stagnalis. Using both in situ and in vitro preparations, glutamate or glutamate agonists produced a depolarization in identified neurons right pedal dorsal 1 and right pedal dorsal 2 and 3. However, attempts to block glutamate-evoked responses with glutamate antagonists were unsuccessful. We examined a potential glutamatergic neuron, visceral dorsal 4. Exogenous application of the peptides (GDPFLRFamide and SDPFLRFamide) could mimic the inhibitory, but not the excitatory effects of visceral dorsal 4 on its postsynaptic cells, implying the presence of a second transmitter. We tested the possibility that glutamate is this second neurotransmitter by using excitatory synapses between visceral dorsal 4 and postsynaptic cells right pedal dorsal 2 and 3, right pedal dorsal 1, visceral F group and right parietal B group neurons. Of all the putative neurotransmitters tested, only glutamate had consistent excitatory effects on these postsynaptic cells. Also, the amplitude of the right pedal dorsal 2 and 3 excitatory postsynaptic potentials was reduced in the presence of N-methyl-D-aspartate and other glutamate agonists, suggesting desensitization of the endogenous transmitter receptor. In conclusion, some identified Lymnaea neurons respond to glutamate via a receptor with novel pharmacological properties. Furthermore, a Lymnaea interneuron may employ glutamate as a transmitter at excitatory synapses.