Prior differences in previous trauma exposure primarily drive the observed racial/ethnic differences in posttrauma depression and anxiety following a recent trauma.

BACKGROUND: Racial and ethnic groups in the USA differ in the prevalence of posttraumatic stress disorder (PTSD). Recent research however has not observed consistent racial/ethnic differences in posttraumatic stress in the early aftermath of trauma, suggesting that such differences in chronic PTSD rates may be related to differences in recovery over time. METHODS: As part of the multisite, longitudinal AURORA study, we investigated racial/ethnic differences in PTSD and related outcomes within 3 months after trauma. Participants (n = 930) were recruited from emergency departments across the USA and provided periodic (2 weeks, 8 weeks, and 3 months after trauma) self-report assessments of PTSD, depression, dissociation, anxiety, and resilience. Linear models were completed to investigate racial/ethnic differences in posttraumatic dysfunction with subsequent follow-up models assessing potential effects of prior life stressors. RESULTS: Racial/ethnic groups did not differ in symptoms over time; however, Black participants showed reduced posttraumatic depression and anxiety symptoms overall compared to Hispanic participants and White participants. Racial/ethnic differences were not attenuated after accounting for differences in sociodemographic factors. However, racial/ethnic differences in depression and anxiety were no longer significant after accounting for greater prior trauma exposure and childhood emotional abuse in White participants. CONCLUSIONS: The present findings suggest prior differences in previous trauma exposure partially mediate the observed racial/ethnic differences in posttraumatic depression and anxiety symptoms following a recent trauma. Our findings further demonstrate that racial/ethnic groups show similar rates of symptom recovery over time. Future work utilizing longer time-scale data is needed to elucidate potential racial/ethnic differences in long-term symptom trajectories.

SARS-CoV-2 Variant Spike and accessory gene mutations alter pathogenesis.

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against SARS-CoV-2, the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the Spike protein. Although these differences in Spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome which may affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other non-spike mutations on SARS-CoV-2 pathogenesis, we synthesized viruses where the WA1 Spike is replaced by each variant spike genes in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to the full variant viruses. Our work has revealed that non-spike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host and can lead to attenuating phenotypes in circulating variants of concern. This work suggests that while Spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may lead to less clinical disease, extended time toward knowing an infection exists in a person and thus increased time for transmission to occur. Significance: A hallmark of the COVID19 pandemic has been the emergence of SARS-CoV-2 variants that have increased transmission and immune evasion. Each variant has a set of mutations that can be tracked by sequencing but little is known about their affect on pathogenesis. In this work we first identify accessory genes that are responsible for pathogenesis in vivo as well as identify the role of variant spike genes on replication and disease in mice. Isolating the role of Spike mutations in variants identifies the non-Spike mutations as key drivers of disease for each variant leading to the hypothesis that viral fitness depends on balancing increased Spike binding and immuno-evasion with attenuating phenotypes in other genes in the SARS-CoV-2 genome.

An outbreak of norovirus gastroenteritis associated with wedding cakes.

We sought to determine the source of a norovirus outbreak among attendees of 46 weddings taking place during a single weekend. Norovirus-compatible illness was experienced by 332 (39%) of wedding guests surveyed; the outbreak affected up to 2700 persons. Illness was associated with eating wedding cake provided by a bakery common to the weddings (adjusted RR 4.5, P<0.001). A cake requiring direct hand contact during its preparation accounted for the majority of illness. At least two bakery employees experienced norovirus-compatible illness during the week preceding the weddings. Identical sequence types of norovirus were detected in stool specimens submitted by two wedding guests, a wedding hall employee, and one of the ill bakery employees. It is likely that one or more food workers at the bakery contaminated the wedding cakes through direct and indirect contact. These findings reinforce the necessity of proper food-handling practices and of policies that discourage food handlers from working while ill.

Crystal structure of human cathepsin V.

Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin V's activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin V's active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.

Violence prevention in the emergency department: clinician attitudes and limitations.

OBJECTIVE: To assess emergency department (ED) clinicians' attitudes and behaviors regarding identification, assessment, and intervention for youth at risk for violence in the ED. DESIGN: Anonymous, cross-sectional written questionnaire. SETTING: The EDs of 3 urban hospitals. SUBJECTS: Emergency medicine residents and faculty, pediatric residents, pediatric emergency medicine fellows and faculty, and ED nurses. RESULTS: A total of 184 (88%) of 208 clinicians completed the questionnaire. Only 15% correctly recognized the lack of existing protocols for addressing youth violence. Clinicians reported being most active in identification of at-risk youth (93% asking context of injury and 82% determining relationships of victim and perpetrator), with pediatricians being more active than general ED clinicians (87% vs 68%; P<.01). Clinicians less often reported performing assessments or referrals of at-risk youth. Nurses and physicians were no different in their reported identification, assessment, or referral behaviors. Barriers identified include concern over upsetting family members, lack of time or skills, and concern for personal safety. Additional clinician training, information about community resources, and specially trained on-site staff were noted by respondents as potential solutions. CONCLUSIONS: Emergency department clinicians recognize the need for evaluation of youth at risk for violence. They are able to identify violently injured youth, but less often perform risk assessment to guide patients to appropriate follow-up resources. Further investigation should address clinician barriers to the complete care of violently injured youth in the ED.

The lysosomal cysteine proteases.

A significant number of exciting papain-like cysteine protease structures have been determined by crystallographic methods over the last several years. This trove of data allows for an analysis of the structural features that empower these molecules as they efficiently carry out their specialized tasks. Although the structure of the paradigm for the family, papain, has been known for twenty years, recent efforts have reaped several structures of specialized mammalian enzymes. This review first covers the commonalities of architecture and purpose of the papain-like cysteine proteases. From that broad platform, each of the lysosomal enzymes for which there is an X-ray structure (or structures) is then examined to gain an understanding of what structural features are used to customize specificity and activity. Structure-based design of inhibitors to control pathological cysteine protease activity will also be addressed.

Mothers with histories of domestic violence in a pediatric emergency department.

OBJECTIVE: To determine the prevalence of domestic violence against mothers in a pediatric emergency department and the relationship of their children to the abusers. DESIGN: Cross-sectional survey of a convenience sample of mothers seeking treatment for their children. SETTING: An urban pediatric emergency department. PARTICIPANTS: A total of 157 mothers with children <3 years of age. Women were excluded if older children or partners were present. RESULTS: A total of 52% of women reported histories of adult physical abuse, 21% reported adult sexual abuse, and 28% reported childhood sexual abuse. A total of 10% of women were in abusive relationships in the past year. Victims of adult physical abuse were more likely to report histories of adult sexual abuse (relative risk [RR]: 4.93) or childhood sexual abuse (RR: 3.13). Intimate partners perpetrated 67% of physical abuse and 55% of sexual abuse. Relatives perpetrated 66% of childhood sexual abuse. Women who revealed histories of childhood sexual abuse were more likely to report adult sexual abuse (RR: 4. 93). A total of 40% of the perpetrators of adult physical abuse, 73% of the perpetrators of past year physical abuse, and 10% of the perpetrators of adult sexual abuse had regular contact with their victims' children. Health care providers screened only 21% of the women for past violence. Victims of domestic violence were no more likely to have been screened than those without histories of physical or sexual abuse. CONCLUSIONS: Mothers of young patients in a pediatric emergency department are often victims of domestic violence. Perpetrators are often close relatives and thus place the victims' children at risk for abuse and for the psychological trauma of witnessing violence. Given the prevalence of domestic violence, families may benefit from routine violence screening and interventions in pediatric emergency departments.

Crystal structure of human cathepsin S.

We have determined the 2.5 A structure (Rcryst = 20.5%, Rfree = 28.5%) of a complex between human cathepsin S and the potent, irreversible inhibitor 4-morpholinecarbonyl-Phe-hPhe-vinyl sulfone-phenyl. Noncrystallographic symmetry averaging and other density modification techniques were used to improve electron density maps which were nonoptimal due to systematically incomplete data. Methods that reduce the number of parameters were implemented for refinement. The refined structure shows cathepsin S to be similar to related cysteine proteases such as papain and cathepsins K and L. As expected, the covalently-bound inhibitor is attached to the enzyme at Cys 25, and enzyme binding subsites S3-S1' are occupied by the respective inhibitor substituents. A somewhat larger S2 pocket than what is found in similar enzymes is consistent with the broader specificity of cathepsin S at this site, while Lys 61 in the S3 site may offer opportunities for selective inhibition of this enzyme. The presence of Arg 137 in the S1' pocket, and proximal to Cys 25 may have implications not only for substrate specificity C-terminal to the scissile bond, but also for catalysis.

Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity.

Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36-59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.

A prevalence survey of abuse and screening for abuse in urgent care patients.

OBJECTIVE: To determine the prevalence of physical and sexual abuse in pregnant and nonpregnant women in an urgent care obstetrics and gynecology triage unit and the frequency with which these patients recall being screened by their health care provider. METHODS: We carried out a structured survey of 255 pregnant and 142 nonpregnant women presenting to an urban New England urgent care obstetrics and gynecology unit between February 1995 and September 1995. Patients in advanced stages of labor or unable to participate due to a language barrier were excluded. The survey consisted of 22 questions, seven of which were modified from the abuse assessment screen. RESULTS: Among 397 participants with complete data, we found that 184 (46%) reported a history of physical or sexual abuse in the past, and 38 (10%) reported recent abuse. Young age and insurance status (Medicaid or uninsured) were associated significantly with recent abuse after we controlled for race, education, and pregnancy status. Only 18% of women recalled being asked about abuse by a health care provider. Young women were more likely to report being asked about abuse. Among women reporting recent abuse, white women were significantly more likely to report being asked about abuse than nonwhite women (P=.02). The majority of women reporting a history of abuse did not recall being screened for violence by a health care provider. CONCLUSION: Women of all ages, income, and ethnic backgrounds reported a history of domestic violence or sexual assault. Providers should incorporate routine screening into the assessment of all women.

Crystal structure of phenylmethanesulfonyl fluoride-treated human chymase at 1.9 A.

The X-ray crystal structure of human chymase has been determined to 1.9 A resolution using molecular replacement methods. This first structure of human chymase is present as the Ser 195 ester of alpha-toluenesulfonic acid. The refined structure (Rcryst = 0.183) shows that the inhibitor phenyl moiety lies at the top of the major specificity pocket, S1, while the sulfur is covalently linked to Ser 195-O gamma. The sulfonyl oxygens interact with the oxyanion hole and with His 57-N delta 1. The presence of the inhibitor disturbs the usual gauche position of His 57 and forces it to the trans conformer. Though the primary binding pockets are similarly specific in chymase and chymotrypsin, examination of the extended substrate binding sites reveals the structural basis for chymase's greater discrimination in choosing substrates. The larger 30s loop and its proximity to the active site indicates that it contacts substrate residues C-terminal to the scissile bond. Modeling of substrate at the chymase active site suggests that binding energy may be gained by three main-chain hydrogen bonds provided by substrate residues P2' and P4' and that discriminating interactions with substrate side chains are also likely. The presence of Lys 40 in S1' of human chymase explains its preference for Asp/Glu at P1'. Moreover, the cationic nature of S1' provides a structural basis for human chymase's poor catalytic efficiency when angiotensin II is the substrate.

Production of crystallizable human chymase from a Bacillus subtilis system.

A Bacillus subtilis strain deficient in seven extracellular proteases was used to produce human mast cell chymase and is a viable expression system for serine proteases and other classes of proteins. Chymase is produced at 0.3-0.5 mg/l and is purified by three chromatography steps. Two crystal forms of PMSF-treated chymase were optimized. The first is C2 with a=47.94 A, b=85.23 A, c=174.18 A, beta=96.74 degrees, and diffracts to at least 2.1 A, while the second is P212121, with cell dimensions a=43.93 A, b=58.16 A, and c=86.09 A, and a diffraction limit of approximately 1.9 A. The first crystal form has either three or four molecules/asymmetric unit, while the second has one molecule/asymmetric unit.

Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex.

Cathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling. To facilitate structural studies of the protein, the wild-type sequence of the protease has been mutated so as to replace a potential N-glycosylation site. We have expressed the mutant human cathepsin K to 190 mg/5 L using the Pichia pastoris expression system. Cathepsin K was inactivated with the mechanism-based inhibitor, APC3328, and crystallized from magnesium formate. A 2.2 A X-ray data set has been collected on crystals belonging to space group P2(1)2(1)2(1), with a = 41.66 A, b = 51.41 A, and c = 107.72 A. There is most likely one molecule per asymmetric unit.

Violence against women: provider barriers to intervention in emergency departments.

OBJECTIVE: To determine: 1) provider behavior in screening for domestic violence (DV) and sexual assault (SA); 2) provider training in DV and SA; 3) provider knowledge of available protocols for DV and SA; and 4) provider perception of barriers to intervention. METHODS: Anonymous, structured surveys were distributed to physicians, nurses, and social workers at an adult ED trauma center, an affiliated pediatric ED, and a women's urgent care center between July and September 1995. RESULTS: Of 207 staff members (59%) responding, 54% and 68% indicated that they never/rarely screen for DV or SA, respectively. Thirty-five percent had received no DV training and 27% had received no SA training. Thirty-one percent of the staff had knowledge of existing protocols for DV and 63% had knowledge of existing protocols for SA. Providers trained in DV were more likely to screen for DV (RR 1.5, 95% CI 1.27-1.92, p < or = 0.001) and SA (RR 1.49, 95% CI 1.24-1.79, p < or = 0.0018), and providers trained in SA were more likely to screen for SA (RR 1.32, 95% CI 1.13-1.54, p = 0.0019) and DV (RR 1.35, 95% CI 1.13-1.60, p = 0.0007). Barriers that the majority of staff experienced in the care of DV/SA victims included: frustration that the victim would return to an abusive partner, concerns about misdiagnosis, lack of time, personal discomfort, reluctance to intrude into familial privacy, and lack of 24-hour social service support. CONCLUSION: Providers surveyed had received little training in and rarely screen for violence, and there are a range of personal and institutional barriers impeding intervention with victims of SA and DV. Institutional changes to enhance training and support providers working in the front line of this epidemic may improve services for victims of violence.

Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons.

The molecular mechanisms underlying the establishment of a postsynaptic receptor mosaic on CNS neurons are poorly understood. One protein thought to be involved is gephyrin, a peripheral membrane protein that binds to the inhibitory glycine receptor and functions in clustering this receptor at synapses in cultured rat spinal cord neurons. We investigated the possible association of gephyrin with synapses in cultured rat hippocampal neurons, where glutamate and GABA but not glycine are the principal transmitters. Gephyrin immunoreactivity was detected in axons as well as dendrites, changing from a predominantly axonal to a more dendritic distribution with time in culture. Gephyrin staining was not distributed uniformly, but always took the form of clusters. Small clusters of gephyrin (0.2 microns 2), present throughout development, were distributed widely and not restricted to synaptic sites. Larger clusters of gephyrin (0.4-10.0 microns 2, sometimes composed of groups of small clusters), which developed in older cells, were localized to a subset of contacts between axons and dendrites. These large clusters were not present at glutamatergic synapses (marked by immunostaining for GluR1), but were closely associated with GABAergic synapses (marked by immunostaining for GABA and glutamic acid decarboxylase). These results, together with previous findings, suggest that gephyrin may function to anchor GABA and glycine receptors, but not glutamate receptors, at postsynaptic sites on central neurons. They also raise the possibility that gephyrin has additional functions, independent of its role at synapses.

High level expression and crystallization of recombinant human cathepsin S.

We have expressed active human cathepsin S to 60 mg/L in Sf9 cells using a baculovirus system. Production of milligram quantities has facilitated crystallographic studies to determine the structure of this enzyme, which has unique properties among lysosomal cysteine proteinases. Recombinant, irreversibly inhibited cathepsin S was crystallized from ammonium phosphate at 17 degrees C. The crystals diffract to at least 2.3 A, and belong to the orthorhombic crystal system with a primitive lattice. Approximate cell dimensions are: a = 37.7 A, b = 73.9 A, and c = 106.7 A. There is most likely one molecule per asymmetric unit.

A structural role for hormone in the thyroid hormone receptor.

The crystal structure of the rat alpha 1 thyroid hormone receptor ligand-binding domain bound with a thyroid hormone agonist reveals that ligand is completely buried within the domain as part of the hydrophobic core. In addition, the carboxy-terminal activation domain forms an amphipathic helix, with its hydrophobic face constituting part of the hormone binding cavity. These observations suggest a structural role for ligand, in establishing the active conformation of the receptor, that is likely to underlie hormonal regulation of gene expression for the nuclear receptors.

The cysteine protease of Trypanosoma cruzi as a model for antiparasite drug design.

By combining new technology in molecular biology, X-ray crystallography, computer graphics and biochemistry, structure-based drug design provides a parallel and cost-effective strategy for identification of new antiparasite chemotherapy. James McKerrow, Mary McGrath and Juan Engel here discuss an example of the amplication of this strategy is its use in targeting the major cysteine protease in Trypanosoma cruzi. Tools from molecular biology helped overcome the obstacle of limited parasite material to allow production of reagent quantities of enzyme for inhibitor screening. Computer graphics analysis and X-ray crystallography are allowing rapid identification of new inhibitors based on either leads already identified or compounds selected by computer graphics screening of chemical databases.

The crystal structure of cruzain: a therapeutic target for Chagas' disease.

Trypanosoma cruzi, a protozoan parasite, is the etiologic agent of American trypanosomiasis or Chagas' disease. Chagas' disease afflicts more than 24 million individuals in South and Central America producing a debilitating life-long disease. It is the leading cause of heart failure in many Latin American countries. Currently, there is no satisfactory treatment for this parasitic infection. Cruzain (also known as cruzipain, gp 57/51), the major cysteine protease present in T. cruzi, is critical for the development and survival of the parasite within the host cells, making this enzyme a target for potential trypanocidal drugs. Here we report the X-ray crystal structure of cruzain complexed with the potent inhibitor Z-Phe-Ala-fluoromethyl ketone. The structure was determined at 2.35 A (Rcryst = 0.15) by molecular replacement using a modified papain as the search model. The refined structure is compared to papain. Features which distinguish cruzain from papain are discussed since they may aid in the design of specificity inhibitors. Fluorescence microscopy shows that a biotinylated form of the bound inhibitor does not effectively reach host proteases in their lysosomal compartment, but is selectively taken up by the parasite. The inhibitor greatly reduces parasitemia in a cell culture system, without adverse effects to mammalian cells. This biological selectivity can be exploited, in conjunction with unique active site features revealed by the crystal structure, to develop chemotherapy for Chagas' disease.