Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli.

BACKGROUND: Escherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don't require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody's disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing. RESULTS: In this study, we found that the reduction in antibody's disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction and free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in E. coli produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction. CONCLUSIONS: During E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction and free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.

Hepatitis B vaccine and risk of acute myocardial infarction among individuals with diabetes mellitus.

PURPOSE: A pre-licensure clinical trial of a two-dose cytosine phosphoguanine adjuvanted hepatitis B vaccine (HEPLISAV-B® [Dynavax, USA]; HepB-CpG vaccine) found an unanticipated numerical imbalance in acute myocardial infarction (AMI) compared to recipients of a three-dose aluminum adjuvanted hepatitis B vaccine (ENGERIX-B® [GlaxoSmithKline, Belgium]; HepB-alum vaccine). A post-licensure study was required to compare AMI rates among recipients of HepB-CpG vaccine and HepB-alum vaccine. Individuals with diabetes mellitus (DM), who are at higher risk of AMI, comprise more than half of the post-licensure study cohort. To inform the ongoing post-licensure study, we examined the association between AMI and receipt of HepB-alum vaccine in individuals with DM. METHODS: We conducted a case-control study nested in a cohort of individuals with DM ages ≥40 years at Kaiser Permanente Southern California using electronic health records. AMI cases from 2012 to 2017 were identified by principal discharge diagnosis and matched 1:1 with randomly selected controls. The adjusted odds ratio (aOR) for receipt of ≥1 HepB-alum vaccine dose was compared for AMI cases and controls using conditional logistic regression. We subsequently performed the same matched case-control analysis stratified by year. RESULTS: Of 8138 matched case-control pairs, 17.4% of cases and 15.0% of controls received HepB-alum vaccine. The aOR of HepB-alum vaccination comparing cases and controls was 0.97 (95% confidence interval 0.87-1.08). Similarly, there was no significant association between HepB-alum vaccine and AMI in any of the study years. CONCLUSIONS: HepB-alum vaccination was not associated with AMI in individuals with DM. This finding will provide contextual insight for the ongoing post-licensure study of HepB-CpG vaccine.

Variation in SARS-CoV-2 molecular test sensitivity by specimen types in a large sample of emergency department patients.

BACKGROUND: Provider-collected nasopharyngeal specimens for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular testing are the standard of care in many clinical settings, but patient-collected saliva and anterior nares specimens are less invasive and more flexible alternatives. Prior studies comparing specimen types for SARS-CoV-2 molecular testing have been limited by small sample sizes and low pretest probability. We conducted a large observational study among symptomatic adults at 7 emergency departments of Kaiser Permanente Southern California to examine sensitivity of SARS-CoV-2 molecular tests by specimen type and patient characteristics. METHODS: Provider-collected nasopharyngeal/oropharyngeal (NP/OP) specimens and patient-collected saliva and anterior nares specimens were collected at the same visit and analyzed with the Roche cobas® SARS-CoV-2 assay. Patients were considered truly positive for SARS-CoV-2 if any of the three specimens was positive and negative if all three specimens were negative. Factors associated with discordant and missed positive results were examined with multivariable logistic regression. RESULTS: Of 2112 patients, 350 (16.6%) were positive for SARS-CoV-2. Sensitivity of NP/OP was 93.7% (95% confidence interval [CI] 90.6%-96.0%), sensitivity of saliva was 87.7% (83.8%-91.0%), and sensitivity of anterior nares was 85.4% (81.3%-89.0%). Patients ages 18-39 years versus ≥40 years were more likely to have discordant results [adjusted odds ratio (aOR) 1.97 (1.12-3.45)], as were patients with <4 symptoms versus ≥4 [aOR 2.43 (1.39-4.25)]. Cycle threshold values were higher for saliva and anterior nares than NP/OP specimens, as well as for specimens in discordant versus concordant sets and patients with fewer symptoms. CONCLUSION: This study provides robust evidence that patient-collected saliva and anterior nares are sensitive for SARS-CoV-2 molecular testing in emergency department settings, particularly among adults ages ≥40 years and those with multiple symptoms. Higher sensitivity of provider-collected NP/OP specimens must be weighed against the benefits of patient-collected specimens in tailored strategies for SARS-CoV-2 testing.

Factors Affecting Microbial Contamination on the Back Surface of Worn Soft Contact Lenses.

SIGNIFICANCE: The results of this study demonstrate that Smart Touch Technology packaging, which is designed to reduce and simplify contact lens handling before insertion, is effective in reducing the frequency of bacterial contamination of the back surface of contact lenses after short-term wear. PURPOSE: The purpose of this study was to investigate the effect of lens packaging type, chelating agent, and finger contamination on microbial contamination on the back surface of worn soft contact lenses. METHODS: Twenty-five subjects completed each contralateral lens wear comparison in this randomized study: Smart Touch Technology versus conventional blister packaging for (1) silicone hydrogel lenses with ethylenediaminetetraacetic acid (EDTA) and (2) hydrogel lenses without EDTA in the packaging, and (3) silicone hydrogel lenses without EDTA versus hydrogel lenses with EDTA both in Smart Touch Technology packaging. Participants washed hands, underwent finger swabs, and inserted the lenses. After 45 minutes, lenses were removed aseptically and the posterior lens surfaces cultured. RESULTS: Thirty-eight subjects (average age, 30.9 ± 12.5 years) participated in this study. Overall, the level of back surface contamination was low for both lens materials, ranging from 0 to 43 colony-forming unit (CFU)/lens for the silicone hydrogel and 0 to 17 CFU/lens for the hydrogel lenses. The proportion of lenses with zero back surface contamination ranged from 16 to 64% for silicone hydrogel lenses and 28 to 64% for hydrogel lenses. Contact lenses from conventional packaging containing EDTA had 3.38 times increased risk (95% confidence interval [CI], 1.02 to 11.11; P = .05) of contamination being present compared with lenses from Smart Touch packaging with EDTA. Contact lenses from conventional packaging without EDTA had 3.4 times increased risk (95% CI, 1.02 to 11.36; P = .05) of contamination being present compared with Smart Touch packaging without EDTA, and silicone hydrogel lenses had a 6.28 times increased risk (95% CI, 1.65 to 23.81; P = .007) of contamination being present compared with hydrogels. The median (interquartile range) number of bacteria isolated from fingers used to perform lens insertion after handwashing but before lens insertion was not significantly different between the silicone hydrogel and hydrogel lenses (63.7 [204.2] vs. 59 [84.5], P = .09). Finger contamination was not significantly associated with lens contamination in the presence or absence of EDTA. CONCLUSIONS: Smart Touch Technology packaging was effective in reducing the proportion of contaminated lenses. Although silicone hydrogel lenses were more likely to be contaminated, the presence of EDTA ameliorated this effect. Finger contamination was not associated with lens contamination.

Computerized Exposure Therapy for Spider Phobia: Effects of Cardiac Timing and Interoceptive Ability on Subjective and Behavioral Outcomes.

OBJECTIVE: Spider phobia is a common form of anxiety disorder for which exposure therapy is an effective first-line treatment. Motivated by the observed modulation of threat processing by afferent cardiac signals, we tested the hypothesis that interoceptive information concerning cardiovascular arousal can influence the outcomes of computerized exposure therapy for spider phobia. METHOD: Fifty-three normal healthy participants with high spider phobia scores underwent one of the following three modified computerized exposure protocols, defined by the timing of exposure to brief spider stimuli within the cardiac cycle: systole (during afferent baroreceptor firing); diastole (during baroreceptor-quiescent interbeat interval); random (noncontingent on cardiac cycle). Outcomes were judged on phobic and anxiety measures and physiological data (skin conductance). Individuals were also rated on interoceptive accuracy. RESULTS: MANCOVA analysis showed that timing group affected the outcome measures (F(10,80) = 2.405, p = .015) and there was a group interaction with interoception ability (F(15,110) = 1.808, p = .045). Subjective symptom reduction was greatest in the systolic group relative to the other two groups (diastolic (t = 3.115, ptukey = .009); random (t = 2.438, ptukey = .048)), with greatest reductions in those participants with lower interoceptive accuracy. Behavioral aversion reduced more in cardiac-contingent groups than the noncontingent (random) group (diastolic (t = 3.295, ptukey = .005); systolic (t = 2.602, ptukey = .032)). Physiological (skin conductance response) responses remained strongest for spider stimuli presented at cardiac systole. CONCLUSIONS: Interoceptive information influences exposure benefit. The reduction in the subjective expression of fear/phobia is facilitated by "bottom-up" afferent signals, whereas improvement in the behavioral expression is further dependent on "top-down" representation of self-related physiology (heart rhythm). Individual interoceptive differences moderate these effects, suggesting means to personalize therapy.

Disparities in mortality for chronic liver disease among Asian subpopulations in the United States from 2007 to 2016.

The Asian American population is characterized by remarkable diversity. Studying Asians as an aggregate group may obscure clinically meaningful heterogeneity. We performed a population-based study using data from the US National Vital Statistics System. We determined the trends in age-standardized mortality rates for chronic liver disease stratified by aetiology among the most populous US-based Asian subgroups (Asian Indians, Chinese, Filipino, Japanese, Korean and Vietnamese) and compared it to non-Hispanic whites. Annual percentage change was calculated to determine temporal mortality patterns using joinpoint analysis. Hepatitis C virus-related mortality rates were higher in non-Hispanic whites compared to individual Asian subgroups, but a sharp decline in mortality rates was noted in 2014 among non-Hispanic whites and all Asian subgroups. Age-standardized hepatitis B virus-related mortality rates were higher in all Asian subgroups as compared to non-Hispanic whites in 2016, with the highest mortality among Vietnamese followed by Chinese. Mortality rates for alcoholic liver disease have been steadily trending upwards in all Asian subgroups, with the highest mortality in Japanese. Overall, age-standardized cirrhosis-related mortality rates were highest in non-Hispanic whites, followed by Japanese, and more distantly by Vietnamese and other subgroups. However, hepatocellular carcinoma-related mortality rates were higher in most Asian subgroups led by Vietnamese, Japanese and Koreans compared to non-Hispanic whites. In this population-based study utilizing a nationally representative database, we demonstrated a marked heterogeneity in the mortality rates of aetiology-specific chronic liver disease among Asian subgroups in the United States.

Clinical Outcomes and Contact Lens Case Contamination Using a Povidone-Iodine Disinfection System.

OBJECTIVE: To assess the incidence of adverse events during the use of a povidone-iodine disinfecting solution (cleadew) and the microbial contamination in contact lens cases. METHODS: A prospective, single-center, open-label, controlled study evaluating the use of cleadew cleaning and disinfecting system in existing daily wear soft contact lens wearers over a 3-month period was conducted. Ocular signs and symptoms during lens wear were assessed at baseline and after 1 and 3 months of using cleadew. Contact lens cases were assessed for the frequency of microbial contamination and the types of microbes, using traditional microbial culture, followed by identification of bacteria using 16S rRNA sequencing. RESULTS: Use of cleadew was well tolerated. There was reduction in corneal staining (0.5±0.5 at 3 months of use) compared with the participant's habitual multipurpose disinfecting system (1.1±1.0); all other clinical signs were not significantly different. There were no cases of solution-induced corneal staining. There was a low adverse event rate of 0.8% per 100 participant-months. Contact lens case contamination was low, with 30% of cases having no culturable microbes. Comparison with previously published data showed that use of cleadew resulted in low frequencies of Gram-positive (49%) and fungal (8%) contamination and a low, but higher than some other disinfecting solutions, level of Gram-negative bacteria. CONCLUSIONS: Cleadew cleaning and disinfecting system was associated with low levels of adverse events during use. Contact lens cases were significantly less frequently contaminated than for some other types of disinfecting solutions.

Genetic prion disease: Experience of a rapidly progressive dementia center in the United States and a review of the literature.

Although prion diseases are generally thought to present as rapidly progressive dementias with survival of only a few months, the phenotypic spectrum for genetic prion diseases (gPrDs) is much broader. The majority have a rapid decline with short survival, but many patients with gPrDs present as slowly progressive ataxic or parkinsonian disorders with progression over a few to several years. A few very rare mutations even present as neuropsychiatric disorders, sometimes with systemic symptoms such as gastrointestinal disorders and neuropathy, progressing over years to decades. gPrDs are caused by mutations in the prion protein gene (PRNP), and have been historically classified based on their clinicopathological features as genetic Jakob-Creutzfeldt disease (gJCD), Gerstmann-Sträussler-Scheinker (GSS), or Fatal Familial Insomnia (FFI). Mutations in PRNP can be missense, nonsense, and octapeptide repeat insertions or a deletion, and present with diverse clinical features, sensitivities of ancillary testing, and neuropathological findings. We present the UCSF gPrD cohort, including 129 symptomatic patients referred to and/or seen at UCSF between 2001 and 2016, and compare the clinical features of the gPrDs from 22 mutations identified in our cohort with data from the literature, as well as perform a literature review on most other mutations not represented in our cohort. E200K is the most common mutation worldwide, is associated with gJCD, and was the most common in the UCSF cohort. Among the GSS-associated mutations, P102L is the most commonly reported and was also the most common at UCSF. We also had several octapeptide repeat insertions (OPRI), a rare nonsense mutation (Q160X), and three novel mutations (K194E, E200G, and A224V) in our UCSF cohort. © 2016 Wiley Periodicals, Inc.

Global Host Partner Perspectives: Utilizing a Conceptual Model to Strengthen Collaboration with Host Partners for International Nursing Student Placements.

OBJECTIVES: Collaboration in international nursing student placements requires equitable partnerships between global partners to address areas of shared importance, such as equity and justice in health promotion. This qualitative study was the first to use the Leffers and Mitchell Conceptual Model for Partnership and Sustainability in Global Health to elicit global host partners' perspectives regarding effective collaboration for Canadian community health nursing placements in the Dominican Republic. DESIGN AND SAMPLE: Focus group and semi-structured interview methodology was conducted with Dominican Republic (Dominican and Haitian) host partners (n = 23) about the engagement processes and host partner factors for effective partnership. RESULTS: Dominican (83%) and Haitian (17%) participants, comprised similar numbers of male and female adults aged 18-60 years (mean age = 36 years), represented the full range of the Dominican Republic host partners (e.g., teachers, health professionals). Interpretive analysis revealed themes that included (1) the unique role of the cultural broker; (2) relational collaboration in a collective society; (3) reciprocal approaches that honor local expertise; and (4) contextual socioeconomic and cultural factors that influence partnerships. CONCLUSIONS: Future research and implications at the individual, community, and policy levels are discussed.

Racial Disparity in HCV Demographics and Treatment Between Interferon Era (2002-2003) and Direct Acting Anti-viral Era (2019).

Introduction  Direct-acting antiviral (DAA) treatment increased the sustained viral response (SVR) rate of patients with the hepatitis C virus (HCV) and eliminated response disparities between African American (AA) and non-AA patients seen with interferon (IFN). The aim of this study was to compare 2019 HCV patients (DAA era) to patients from January 1, 2002 and December 31, 2003 (IFN era) in our predominantly AA clinic population. Methods We extracted data on 585 HCV patients seen in 2019 (DAA era) and compared them to 402 patients seen in the IFN era. Results Most HCV patients were born between 1945 and 1965, but in the DAA era more younger patients were identified. Non-AA patients in both eras were less likely to be infected with genotype 1 compared to AA (95% vs 54%, P<0.001). Fibrosis was not increased in the DAA Era as compared to the IFN era as assessed either by serum-based assays (APRI, FIB-4) or transient elastography (FibroScan) (DAA era) vs biopsy (IFN era). More patients were treated in 2019 compared to 2002-2003 (159/585=27% vs 5/402=1%). For untreated patients, subsequent treatment within one year of the initial visit was low and similar in both eras (35%). Conclusion There continues to be a need to screen patients born between 1945 and 1965 for HCV as well as to identify increasing numbers of patients below this age cohort. Even though current therapies are oral, highly effective, and can be 8-12 weeks in duration, significant numbers of patients were not treated within a year of first visit.

Clinical overlap between Jakob-Creutzfeldt disease and Lewy body disease.

OBJECTIVE: Sporadic Jakob-Creutzfeldt disease (sCJD) and dementia with Lewy bodies (DLB) have overlapping clinical symptoms that can lead to their misdiagnosis. We delineated the clinical overlap between sCJD and DLB, and assessed the value of magnetic resonance imaging (MRI) to differentiate between them. METHODS: Medical records, MRI, electroencephalogram (EEG) and cerebrospinal fluid (CSF) were reviewed from 56 sCJD and 30 DLB subjects. RESULTS: 46% of sCJD subjects met probable DLB criteria and 40% of DLB subjects met probable CJD criteria. A greater proportion of sCJD subjects had cerebellar signs (66% vs. 10%, p<0.001), myoclonus (64% vs. 30%, p=0.002), and visual symptoms (other than hallucinations) (61% vs. 7%, p<0.001), whereas more DLB subjects had hallucinations (70% vs. 39%, p=0.007) and fluctuations (57% vs. 23%, p=0.002). Cortical and/or basal ganglia MRI diffusion weighted imaging hyperintensities consistent with sCJD were seen in 96% of sCJD subjects but in none with DLB. Logistic regression in sCJD revealed that those meeting probable DLB criteria were more likely to have occipital lobe involvement on MRI (OR 1.4, p=0.058, model p=0.022). Parietal lobe involvement on MRI was a predictor of "Other Focal Cortical signs" (OR 1.9, p=0.021). EEG and CSF assessments lacked sensitivity for sCJD as 48% of sCJD patients had a negative EEG; 67% of the 36 sCJD patents with a CSF evaluation had a negative or inconclusive 14-3-3 result. Too few DLB patients had EEG or CSF to assess their utility. CONCLUSION: Sporadic CJD and DLB have significant symptom overlap. MRI helps differentiate these diseases and is related to the signs/symptoms observed in sCJD.

The effect of hydroxypropyl-guar nanoemulsion on signs and symptoms of dry eye.

PURPOSE: To examine the effect of hydroxypropyl-guar nanoemulsion (HP-Guar nanoemulsion, Alcon Laboratories Ltd, Fort Worth, TX, USA) versus saline (0.9% sodium chloride; Pfizer Inc., Bentley WA Australia) on the comfort and tear film properties of people with dry eye disease both in the short-term (up to 2 h post-drop instillation) and longer-term (after 4 weeks of 4-times daily use), and to examine the effect on tear inflammatory markers after 4 weeks. METHODS: This was a prospective, investigator-masked, randomised, cross-over dispensing study. Twenty participants with dry eye disease (5 males: 15 females) with average age 46.9 ± 14.4 (range 26 to 70) years were randomized to either HP-Guar nanoemulsion, or saline eye drops. Ocular symptoms, lipid layer thickness, tear evaporation, tear osmolarity and non-invasive break-up time were measured pre-drop instillation, 1 h and 2 h post-eye drop instillation, and after 4-weeks of 4-times daily drop use. Tear inflammatory mediators were measured pre-drop instillation and after 4-weeks. After 4-weeks, patients had a 4-week washout period and then crossed over to the alternate drop. RESULTS: With HP-Guar nanoemulsion, participants reported less grittiness/burning/stinging 1 h post eye drop instillation compared to baseline (79.5 ± 23.3 vs. 66.8 ± 27.7, p = 0.02); less dryness 1 h and 2 h post eye drop instillation compared to baseline (77.8 ± 23.0 and 76.2 ± 23.7 vs. 61.0 ± 27.1 respectively, p < 0.01 for both); and greater overall satisfaction 1 h post drop instillation and after 4 weeks of daily use compared to baseline (80.4 ± 21.6 and 83.4 ± 16.6 vs. 68.6 ± 26.0 respectively, p ≤ 0.011 for both). With saline, participants reported less dryness after 4 weeks of daily use compared to baseline (74.2 ± 23.8 vs. 60.2 ± 24.0, p < 0.01). For HP-Guar nanoemulsion, average lipid layer thickness was significantly thicker 2 h post drop-instillation (79.5 ± 21.7 nm) compared to baseline (63.7 ± 18.9 nm) and the 4-week assessment (62.4 ± 23.1 nm, p < 0.01 for both). For saline, average lipid layer thickness was significantly thicker at 1 h and 2 h post eye drop instillation (76.0 ± 23.8 nm and 80.4 ± 24.8 nm) compared to baseline (61.0 ± 15.6 nm, p < 0.01 for both). There was no difference in inflammatory mediators or other tear variables between drops or visits. CONCLUSION: HP-Guar nanoemulsion was more effective for improving a range of subjective dry eye symptoms both in the short and long-term compared to saline. Both HP-Guar nanoemulsion and saline transiently increased lipid layer thickness.

The enigma of weight: Figures, flux, and fitting in.

Purpose: In Western society, the measurement of weight is prioritized over a person's bodily experience. Hermeneutic philosopher Gadamer warned against the emphasis on measurement, rather than experience, in the medical sciences. An examination of the complexity of the experience of weight provides the opportunity to shift focus from quantifying the connection between health and weight to the experience of the person being weighed. Methods: This qualitative hermeneutic study aims to understand people's experiences of weight from the interviews of professionals (n = 7) and lay experts (n = 10). Interviews were analyzed using an interpretive hermeneutic method. Results: The interviews revealed that weight was experienced as a number imbued with meaning and bias, as a number that could be manipulated, and as a constant and anticipated bodily change. Weight change was expected and often unwelcomed, despite weight being a quality of the body that is always in flux. External measures of weight meant to monitor wellness and health inadvertently became an unhealthy fixation that prevented some participants from fully participating in life events and appreciating the stages their bodies were in. Conclusion: Weight change is a necessary condition of being human, and bodies are and will be constantly changing. To achieve health and harmony, one must fit together the acceptance of change and their bodily experience of weight. It is often the preoccupation with weight, not weight itself, that gets in the way of living.

The optics of weight: expert perspectives from the panopticon and synopticon.

Objective: That we all weigh something is a fact of life, yet the material reality of weight is refracted through multiple layers of surveillance revealing contradictions in experience and understanding, depending on one's vantage point. We explored the complexities of weight with the specific aim of furthering understanding of this multifaceted surveillance.Methods and Measures: We used hermeneutics, the philosophy and practice of interpretation, as the method of inquiry. Ten experts by experience and seven professional experts participated in interviews, which were audio- recorded, transcribed, and analyzed. Interpretations were developed through group discussions among the eight authors and reiterative writing.Results: Using the metaphor of optics, we demonstrate how the interplay of the panopticon (the few watching the many) and synopticon (the many watching the few) help us gain a deeper understanding of weight through "fitting in," being "captured by numbers," "dieting: the tyrannic tower," and "the male gaze."Conclusion: Monitoring and judging body weight have become so normative in Western society that "weight watching" practices are synonymous with good citizenship and moral character. This study offers insight about how weight is conceptualized in personal and professional contexts, with implications for body image, dieting, eating disorders, public health, and weight bias.

The development and initial validation of the Chinese Significant Wish Fulfillment Scale.

BACKGROUND: This paper reports the development and validation of the Chinese Significant Wish Fulfillment Scale (CSWFS), a new multidimensional scale for assessing the perceived importance and level of fulfillment of wishes of older adults. METHODS: Three studies were involved. Study 1 developed a 26-item pool on wish fulfillment through in-depth interviews with 22 older adults. Study 2 reduced the pool to 23 items through validation with a new sample of 315 older adults and examined the internal reliability. Study 3 involved a confirmatory factor analysis (CFA) and examined the test-retest reliability and the convergent validity of the scale with the construct of regret. RESULTS: A five-factor structure model of 23 items was identified through exploratory factor analysis, which accounted for 51.67% of variance. As informed by the CFA in Study 3, a five-factor 22-item model was the best fit. Internal reliability and test-retest reliability was found to be good. Convergent validity was examined through correlation with the construct of future regret. The correlation, though statistically significant, was small. CONCLUSION: CSWFS demonstrates good psychometric properties, but the construct might be slightly different from that of future regrets. Probably, CSWFS addresses a construct that is under-explored but is of importance to older adults and especially to the Chinese community.

High Throughput Detection of 327 Drugs in Blood by LC-MS-MS with Automated Data Processing.

The described procedure provides a rapid technique for the detection and semi-quantitation of a large number of drugs in blood. This procedure uses a minimal sample volume and employs a one-step liquid extraction and automated data processing to yield rapid turnaround times. A total of 327 of the most commonly used medicinal and illicit drugs in Australia were selected including various amphetamines, anesthetics, antidepressants, antipsychotics, anticonvulsants, benzodiazepines, beta blockers, opioid and nonopioid analgesics, stimulants, THC and a large number of synthetic cannabinoids and other novel psychoactive substances. The extracts were subject to 5-minute chromatography using a Kinetex C18 50 × 4.6 mm 2.6 µm solid-core analytical column and analyzed using a Sciex 3200 Q-TRAP MS-MS (+ ESI, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Matrix effects and extraction efficiencies were acceptable with most analytes showing > 80% response and low variation (within 25%RSD). Cannabinoids were most affected by the matrix and yielded poorest recovery values but were still detectable. Precision, accuracy, repeatability and multipoint linearity were assessed for all analytes. The method has been used in routine practice in the forensic toxicology service at the Victorian Institute of Forensic Medicine in over 6000 coronial investigations using both postmortem and clinical blood specimens. This technique has greatly increased throughput, reduced turnaround times and allowed for rapid same-day analysis of results when needed. The method is routinely used in routine overnight testing with results reported to pathologists within 4 h of data acquisition. This rapid toxicological technique is used in conjunction with other investigative processes such as full-body CT imaging, review of case circumstances and medical histories to provide an efficient death investigation process.

Functional Characterization of Ovine Dorsal Root Ganglion Neurons Reveal Peripheral Sensitization after Osteochondral Defect.

Knee joint trauma can cause an osteochondral defect (OD), a risk factor for osteoarthritis (OA) and cause of debilitating pain in patients. Rodent OD models are less translatable because of their smaller joint size and open growth plate. This study proposes sheep as a translationally relevant model to understand the neuronal basis of OD pain. A unilateral 6-mm deep OD was induced in adult female sheep. Two to six weeks after operation, lumbar dorsal root ganglia (DRG) neurons were collected from the contralateral (Ctrl) and OD side of operated sheep. Functional assessment of neuronal excitability and activity of the pain-related ion channels transient receptor potential vanilloid receptor 1 (TRPV1) and P2X3 was conducted using electrophysiology and Ca2+ imaging. Immunohistochemistry was used to verify expression of pain-related proteins. We observed that an increased proportion of OD DRG neurons (sheep, N = 3; Ctrl neurons, n = 15, OD neurons, n = 16) showed spontaneous electrical excitability (Ctrl: 20.33 ± 4.5%; OD: 50 ± 10%; p = 0.009, unpaired t test) and an increased proportion fired a greater number of spikes above baseline in response to application of a TRPV1 agonist (capsaicin) application (Ctrl: 40%; OD: 75%; p = 0.04, χ2 test). Capsaicin also produced Ca2+ influx in an increased proportion of isolated OD DRG neurons (Ctrl: 25%; OD: 44%; p = 0.001, χ2 test). Neither protein expression, nor functionality of the P2X3 ion channel were altered in OD neurons. Overall, we provide evidence of increased excitability of DRG neurons (an important neural correlate of pain) and TRPV1 function in an OD sheep model. Our data show that functional assessment of sheep DRG neurons can provide important insights into the neural basis of OD pain and thus potentially prevent its progression into arthritic pain.

The Arabidopsis Protein Disulfide Isomerase Subfamily M Isoform, PDI9, Localizes to the Endoplasmic Reticulum and Influences Pollen Viability and Proper Formation of the Pollen Exine During Heat Stress.

Plants adapt to heat via thermotolerance pathways in which the activation of protein folding chaperones is essential. In eukaryotes, protein disulfide isomerases (PDIs) facilitate the folding of nascent and misfolded proteins in the secretory pathway by catalyzing the formation and isomerization of disulfide bonds and serving as molecular chaperones. In Arabidopsis, several members of the PDI family are upregulated in response to chemical inducers of the unfolded protein response (UPR), including both members of the non-classical PDI-M subfamily, PDI9 and PDI10. Unlike classical PDIs, which have two catalytic thioredoxin (TRX) domains separated by two non-catalytic TRX-fold domains, PDI-M isoforms are orthologs of mammalian P5/PDIA6 and possess two tandem catalytic domains. Here, PDI9 accumulation was found to be upregulated in pollen in response to heat stress. Histochemical staining of plants harboring the PDI9 and PDI10 promoters fused to the gusA gene indicated they were actively expressed in the anthers of flowers, specifically in the pollen and tapetum. Immunoelectron microscopy revealed that PDI9 localized to the endoplasmic reticulum in root and pollen cells. transfer DNA (T-DNA) insertional mutations in the PDI9 gene disrupted pollen viability and development in plants exposed to heat stress. In particular, the pollen grains of pdi9 mutants exhibited disruptions in the reticulated pattern of the exine and an increased adhesion of pollen grains. Pollen in the pdi10 single mutant did not display similar heat-associated defects, but pdi9 pdi10 double mutants (DMs) completely lost exine reticulation. Interestingly, overexpression of PDI9 partially led to heat-associated defects in the exine. We conclude that PDI9 plays an important role in pollen thermotolerance and exine biogenesis. Its role fits the mechanistic theory of proteostasis in which an ideal balance of PDI isoforms is required in the endoplasmic reticulum (ER) for normal exine formation in plants subjected to heat stress.