ObjectiveTo examine the protective effects of appropriate personal protective equipment for frontline healthcare professionals who provided care for patients with coronavirus disease 2019 (covid-19).DesignCross sectional study.SettingFour hospitals in Wuhan, China.Participants420 healthcare professionals (116 doctors and 304 nurses) who were deployed to Wuhan by two affiliated hospitals of Sun Yat-sen University and Nanfang Hospital of Southern Medical University for 6-8 weeks from 24 January to 7 April 2020. These study participants were provided with appropriate personal protective equipment to deliver healthcare to patients admitted to hospital with covid-19 and were involved in aerosol generating procedures. 77 healthcare professionals with no exposure history to covid-19 and 80 patients who had recovered from covid-19 were recruited to verify the accuracy of antibody testing.Main outcome measuresCovid-19 related symptoms (fever, cough, and dyspnoea) and evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, defined as a positive test for virus specific nucleic acids in nasopharyngeal swabs, or a positive test for IgM or IgG antibodies in the serum samples.ResultsThe average age of study participants was 35.8 years and 68.1% (286/420) were women. These study participants worked 4-6 hour shifts for an average of 5.4 days a week; they worked an average of 16.2 hours each week in intensive care units. All 420 study participants had direct contact with patients with covid-19 and performed at least one aerosol generating procedure. During the deployment period in Wuhan, none of the study participants reported covid-19 related symptoms. When the participants returned home, they all tested negative for SARS-CoV-2 specific nucleic acids and IgM or IgG antibodies (95% confidence interval 0.0 to 0.7%).ConclusionBefore a safe and effective vaccine becomes available, healthcare professionals remain susceptible to covid-19. Despite being at high risk of exposure, study participants were appropriately protected and did not contract infection or develop protective immunity against SARS-CoV-2. Healthcare systems must give priority to the procurement and distribution of personal protective equipment, and provide adequate training to healthcare professionals in its use.

Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features.

No abstract

Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [3H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious “mixing” experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among 5 pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [3H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer.

The COVID-19 outbreak can be seen as a 'big test' for China; a summative assessment of its preparedness on multiple fronts, including medical education. Being intimately involved in the coordinated response, the First Affiliated Hospital of Sun Yat-sen University has been a first-hand witness to the strengths and weaknesses of the current medical education system in China. On the one hand, we believe that the distinguished contributions in disease containment efforts by healthcare professionals indicated that our medical education system has achieved its intended outcomes and is socially accountable. On the other hand, we have also identified three major issues that need to be addressed from an educational standpoint: insufficient emphasis on public health emergency preparedness; unsophisticated mechanisms for interdisciplinary cooperation; and inadequate guidance in medical ethics. Whilst these reflections might be seen in its summative form, we would suggest changing it to that of a formative process, where we learn from our assessment through observation and feedback of the gaps, upon which improvement of our present situation can be made. We hope that these lessons may be helpful to our colleagues in the rest of China and around the world, who are engaged in medical educational reform.

Phosphorylation (P-) of cAMP-response element-binding protein (CREB) by protein kinase A or mitogen-activated protein kinases was implicated in mediating the increased tyrosine hydroxylase (TH) gene expression after prolonged exposure to nicotine in vivo and in cell culture. We examined the time course and signaling pathways for phosphorylation of CREB and possible involvement of ATF-2. Treatment of PC12 cells with 200 M nicotine triggered rapid but transient elevation of P-CREB followed by a second sustained rise after 2-5 h of continuous nicotine. In contrast, ERK1/2 was only phosphorylated with short term nicotine exposure. MEK inhibitor U0126 abolished nicotine-induced rise in P-ERK1/2, but not P-CREB, nor did it inhibit nicotine-evoked elevation in TH promoter activity, indicating that ERK1/2 was not needed for induction of TH gene expression by nicotine. In contrast, protein kinase A inhibitor H-89 or Ca 2؉ / calmodulin-activated protein kinase inhibitor KN-93 reduced the nicotine-triggered rise in P-CREB and TH promoter activity. There was a delayed elevation of P-ATF-2 after 1 h of nicotine treatment, accompanied by increased ATF-2 protein. Upstream kinase JNK, but not p38, was phosphorylated especially after 5 min to 2 h of nicotine exposure. To examine the requirement for CREB and ATF-2, cells were transfected with dominant negative forms of ATF-2 or CREB. Both reduced the basal TH promoter activity and the response to nicotine. Knockdown of ATF-2 or CREB with siRNA did not alter basal TH promoter activity or mRNA but greatly attenuated the response to nicotine. The results suggest that both ATF-2 and CREB mediate activation of TH gene transcription by nicotine.Administration of nicotine in vivo and in cell culture triggers increased expression of a number of genes related to neurosecretion including the catecholamine biosynthetic enzymes. These changes in gene expression may be involved in neurochemical and cardiovascular effects of smoking. Nicotine is a potent sympathomimetic agent, and its administration, in doses similar to those obtained in smoking, increases heart rate and systolic and diastolic blood pressure in humans and many animal species (for review, see Ref. 1). These cardiovascular effects are largely attributed to the direct stimulation of release of catecholamines, epinephrine and norepinephrine, from the adrenal medulla and peripheral sympathetic nerve endings (2-4). In this regard a polymorphism in the gene for tyrosine hydroxylase (TH), 2 the rate-limiting enzyme in catecholamine biosynthesis, has been associated with tobacco use (5).Transcriptional mechanisms are at least partially responsible for the nicotine triggered changes in TH gene expression. The response of TH to nicotine has been proposed to be mediated by CREB. Nicotine treatment both in vivo and in vitro induces phosphorylation of CREB (6 -8).The cAMP/calcium response element (CRE/CaRE) in the TH promoter is required for its transcriptional activation by nicotine in PC12 cells (6). CREB is activated through the phosphor...

J. Neurochem. (2010) 114, 873–885. Abstract Our previous work suggested a role for oligomerization in regulating dopamine transporter (DAT) internalization, with d‐amphetamine dissociating DAT oligomers and monomers being endocytosed. This model was put to detailed testing in the present work with the use of DAT constructs differentially tagged with Myc or Flag, reversal of tags in co‐immunoprecipitation and cross‐linking assays, and application of antibodies against different tags in biotinylation experiments. Upon pairing wild‐type (WT) DAT with W84L mutant, effects of d‐amphetamine on oligomerization (decrease) but not surface DAT are observed. Internalization of W84L monomers appears to be slow as inferred from the inability of d‐amphetamine to reduce surface Myc upon co‐expressing Flag‐WT with Myc‐W84L but not Myc‐WT with Flag‐W84L, and from the sluggish Myc‐W84L endocytosis rate (both with or without d‐amphetamine). Results obtained for D313N, D345N, or D436N mutants can all be accommodated by a model in which d‐amphetamine is unable to dissociate mutant protomers from oligomers (tetramers or higher‐order assemblies) that contain them; this interpretation is confirmed in experiments with both tag reversal in co‐expression and antibody reversal in western blotting. Upon co‐transfecting Myc‐ and Flag‐tagged constructs, resulting tetramers can be calculated to be composed of different species (MycMycMycMyc, MycMycMycFlag, MycMycFlagFlag, MycFlagFlagFlag, and FlagFlagFlagFlag), but it is shown that outcomes predicted by models based on MycMycFlagFlag oligomers are not changed in a major way by the occurrence of the additional species.

Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug.