Rapid publications risk the integrity of science in the era of COVID-19.

BACKGROUND: Preprint manuscripts, rapid publications and opinion pieces have been essential in permitting the lay press and public health authorities to preview data relating to coronavirus disease 2019 (COVID-19), including the range of clinical manifestations and the basic epidemiology early on in the pandemic. However, the rapid dissemination of information has highlighted some issues with communication of scientific results and opinions in this time of heightened sensitivity and global concern. MAIN TEXT: Rapid publication of COVID-19 literature through expedited review, preprint publications and opinion pieces are important resources for the medical scientific community. Yet the risks of unverified information loom large in times when the healthcare community is desperate for information. Information that has not been properly vetted, or opinion pieces without solid evidence, may be used to influence public health policy decisions. We discuss three examples of unverified information and the consequences in this time of high anxiety surrounding COVID-19. CONCLUSIONS: In an era when information can be widely and swiftly disseminated, it is important to ensure that the scientific community is not an inadvertent source of misinformation. This will require a multimodal approach, with buy-in from editors, publishers, preprint servers, authors and journalists. The landscape of medical publications has changed, and a collaborative approach is required to maintain a high standard of scientific communications.

Partial trisomy 3p24.3 and partial monosomy 5p15.33: case report and a literature review.

We report on a preterm neonate with a deletion of the distal short arm of chromosome 5p15.33 and partial trisomy of the distal short arm of chromosome 3p24.3. The patient was the first-born monozygotic twin. There were no pertinent facial or physical features except a small lower lip hemangioma. The neonate presented with cardiac defects, which included a patent ductus arteriosus, an atrial septal defect and ventricular septal defects. After 94 days of age, however, the patient died from superior vena cava syndrome, recurrent chylothoraces and generalized anasarca. Array comparative genomic hybridization (aCGH) using a custom oligonucleotide microarray (Agilent 180,000 probe platform revealed a terminal duplication of 1,128 oligonucleotide probes from 3pter to 3p24.3, spanning approximately 20.4 megabases (Mb), and a terminal deletion of 271 oligonucleotide probes from 5pter to 5p15.33, spanning approximately 4.3 Mb. This is the first report of a patient with partial trisomy 3p24.3 and partial monosomy 5p15.33 without major dysmorphic features.

Holt-Oram syndrome is a genetically heterogeneous disease with one locus mapping to human chromosome 12q.

Holt-Oram syndrome (HOS) is an autosomal dominant condition affecting the heart and upper limbs. We have sought to identify the location of this gene using microsatellite DNA markers in a linkage study. Of seven families analysed, five show linkage between HOS and markers on chromosome 12q. But the two remaining families, phenotypically indistinguishable from the others, do not show this linkage. Analysis with the computer program HOMOG indicates that HOS is a heterogeneous disease. Our analysis places one HOS locus in a 21 cM interval in the distal region of chromosome 12q. The localization of a gene for HOS, reported here, represents an important step towards a better understanding of limb and cardiac development.

Standards for clinical trials for treating TB.

BACKGROUND: The value, speed of completion and robustness of the evidence generated by TB treatment trials could be improved by implementing standards for best practice.METHODS: A global panel of experts participated in a Delphi process, using a 7-point Likert scale to score and revise draft standards until consensus was reached.RESULTS: Eleven standards were defined: Standard 1, high quality data on TB regimens are essential to inform clinical and programmatic management; Standard 2, the research questions addressed by TB trials should be relevant to affected communities, who should be included in all trial stages; Standard 3, trials should make every effort to be as inclusive as possible; Standard 4, the most efficient trial designs should be considered to improve the evidence base as quickly and cost effectively as possible, without compromising quality; Standard 5, trial governance should be in line with accepted good clinical practice; Standard 6, trials should investigate and report strategies that promote optimal engagement in care; Standard 7, where possible, TB trials should include pharmacokinetic and pharmacodynamic components; Standard 8, outcomes should include frequency of disease recurrence and post-treatment sequelae; Standard 9, TB trials should aim to harmonise key outcomes and data structures across studies; Standard 10, TB trials should include biobanking; Standard 11, treatment trials should invest in capacity strengthening of local trial and TB programme staff.CONCLUSION: These standards should improve the efficiency and effectiveness of evidence generation, as well as the translation of research into policy and practice.

Extracorporeal shock wave treatment of non- or delayed union of proximal metatarsal fractures.

BACKGROUND: Nonunion or delayed union of fractures in the proximal aspect of metatarsals 1 to 4 and Zone 2 of the fifth metatarsal were treated by high energy extracorporeal shock wave treatment (ESWT) to study the safety and efficacy of this method of treatment in a FDA study of the Ossatron device. MATERIALS AND METHODS: In a prospective single-arm, multi-center study, 34 fractures were treated in 32 patients (two subjects had two independent fractures) with ESWT. All fractures were at least 10 (range, 10 to 833) weeks after injury, with a median of 23 weeks. ESWT application was conducted using a protocol totaling 2,000 shocks for a total energy application of approximately 0.22 to 0.51 mJ/mm2 per treatment. The mean ESWT application time for each of the treatments was 24.6 +/- 16.6 minutes, and anesthesia time averaged 27.1 +/- 10.4 minutes. All subjects were followed for 1 year after treatment at intervals of 12 weeks, 6, 9, and 12 months. RESULTS: The overall success rate at the 12-week visit was 71% with low complications, significant pain improvement as well as improvement on the SF-36. The success/fail criteria was evaluated again at the 6- and 12-month followup, showing treatment success rates of 89% (23/26) and 90% (18/20), respectively. The most common adverse event was swelling in the foot, reported by five subjects (15.6%). CONCLUSION: High-energy ESWT appears to be effective and safe in patients for treatment of nonunion or a delayed healing of a proximal metatarsal, and in fifth metatarsal fractures in Zone 2.

Fever as a predictor of adverse outcomes in COVID-19.

BACKGROUND/INTRODUCTION: There are little data on outcomes of COVID-19 patients with the presence of fever compared to the presence of symptoms. AIM: We examined the associations between symptomology, presence of fever and outcomes of a COVID-19 cohort. DESIGN AND METHODS: Between 23 January and 30 April 2020, 554 COVID-19 patients were admitted to a tertiary hospital in Singapore. They were allocated into four groups based on symptomology and fever-Group 1: asymptomatic and afebrile, Group 2: symptomatic but afebrile, Group 3: febrile but asymptomatic and Group 4: symptomatic and febrile. The primary outcomes were intensive care unit (ICU) admissions and mortality. The composite end-point included ICU admissions, mortality or any COVID-19 related end-organ involvement. RESULTS: There were differences in ferritin (P=0.003), C-reactive protein (CRP) levels (P<0.001) and lymphopenia (P=0.033) across all groups, with the most favourable biochemical profile in Group 1, and the least in Group 4. Symptomatic groups (Groups 2 and 4) had higher ICU admissions (1.9% and 6.0%, respectively, P=0.003) than asymptomatic groups (Groups 1 and 3). Composite end-point was highest in Group 4 (24.0%), followed by Group 3 (8.6%), Group 2 (4.8%) and Group 1 (2.4%) (P<0.001). The presence of fever (OR 4.096, 95% CI 1.737-9.656, P=0.001) was associated with the composite end-point after adjusting for age, pulse rate, comorbidities, lymphocyte, ferritin and CRP. Presence of symptoms was not associated with the composite end-point. DISCUSSION/CONCLUSION: In this COVID-19 cohort, presence of fever was a predictor of adverse outcomes. This has implications on the management of febrile but asymptomatic COVID-19 patients.

Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro.

In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations greater than 24 mg/liter (2 X 10(-4) M), cysteine was acutely toxic to trypanosome concentrations of 3 X 10(7)/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H2O2 produced by cysteine autoxidation. In uptake studies using [35S]cysteine and [35S]cystine, T. brucei efficiently incorporated only cysteine. The Km for cysteine uptake was 4 X 10(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.

Understanding the optical spectroscopy of amphiphilic molecular rectifiers: a density functional approach.

We present results of first principles density functional theory calculations of the electronic and atomic structural properties of model Z-type Langmuir-Blodgett (LB) layers comprising amphiphilic quinolinium tricyanoquinodimethanide (Q3CNQ) chromophores. We find that the chromophore electronic ground state is not as clearly "zwitterionic" as required by models to explain electrical rectification purportedly seen in such systems. The computed visible region transitions are not what have been assumed to be the intervalence charge transfer bands seen in the visible region of molecules in Z-type LB films. Our own LB deposition and spectroscopic studies suggest that almost all visible region features previously seen may be ascribed to aggregates. The calculated lowest energy electronic excitation between HOMO and LUMO levels, which is located in the near infrared region, has a transition moment aligned approximately 9° off the molecular long axis, and has a normalized oscillator strength of 1 order of magnitude higher than those of the visible region transitions. This most dominant feature has been neglected from discussions of Langmuir-Blodgett layer rectification but our own deposition studies show no sign of this feature, indicating that the structure of the modeled system differs from that of typical experimental structures. The model indicates that such idealized LB layer structures cannot confidently be invoked to explain their experimental optical or electrical properties.

Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system.

Glycosylinositol phospholipid (GPI) membrane anchors are the sole means of membrane attachment of a large number of cell surface proteins, including the variant surface glycoproteins (VSGs) of the parasitic protozoan, Trypanosoma brucei. Biosynthetic data suggest that GPI-anchored proteins are synthesized with carboxy-terminal extensions that are immediately replaced by GPI, suggesting the existence of preformed GPI species available for transfer to the nascent protein in the ER. Candidate precursor glycolipids having a linear sequence indistinguishable from the conserved core structure found on all GPI anchors, have been characterized in T. brucei. In this paper we describe the transfer of three GPI variants to endogenous VSG in vitro. GPI addition is not reduced by inhibitors of protein synthesis and does not require ATP or GTP, consistent with a transpeptidation mechanism.

Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion.

Null mutants of the Trypanosoma cruzi insect stage-specific glycoprotein GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromycin phosphotransferase genes were flanked by GP72 sequences. These plasmids were sequentially transfected into T. cruzi epimastigotes by electroporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No aberrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsewhere in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the normal attachment of the flagellum to the cell membrane of the parasite is lost.

Transport of fluorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum-infected cells.

The asexual development of the human malaria parasite Plasmodium falciparum is largely intraerythrocytic. When 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]caproyl] phosphatidylcholine (NBD-PC) was incorporated into infected and uninfected erythrocyte membranes at 0 degrees C, it remained at the cell surface. At 10 degrees C, the lipid was rapidly internalized in infected erythrocytes at all stages of parasite growth. Our results indicate that the internalization of NDB-PC was not because of endocytosis but rapid transbilayer lipid flip-flop at the infected erythrocyte membrane, followed by monomer diffusion to the parasite. Internalization of the lipid was inhibited by (a) depleting cellular ATP levels; (b) pretreating the cells with N-ethyl maleimide or diethylpyrocarbonate; and (c) 10 mM L-alpha-glycerophosphorylcholine. The evidence suggests protein-mediated and energy dependent transmembrane movement of the PC analogue. The conditions for the internalization of another phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) were distinct from that of NBD-PC and suggest the presence of additional mechanism(s) of parasite-mediated lipid transport in the infected host membrane. In spite of the lack of bulk, constitutive endocytosis at the red cell membrane, the uptake of Lucifer yellow by mature infected cells suggests that microdomains of pinocytotic activity are induced by the intracellular parasite. The results indicate the presence of parasite-induced mechanisms of lipid transport in infected erythrocyte membranes that modify host membrane properties and may have important implications on phospholipid asymmetry in these membranes.

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei.

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.

Expression of a bacterial gene in a trypanosomatid protozoan.

A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control experiments. This system provides a method for defining transcriptional control elements in trypanosomes.

Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei.

The paradigm of antigenic variation in parasites is the variant surface glycoprotein (VSG) of African trypanosomes. Only one VSG is expressed at any time, except for short periods during switching. The reasons for this pattern of expression and the consequences of expressing more than one VSG are unknown. Trypanosoma brucei was genetically manipulated to generate cell lines that expressed two VSGs simultaneously. These VSGs were produced in equal amounts and were homogeneously distributed on the trypanosome surface. The double-expressor cells had similar population doubling times and were as infective as wild-type cells. Thus, the simultaneous expression of two VSGs is not intrinsically harmful.

Estimation of total body water from bioelectrical impedance spectroscopy in oncology outpatients receiving radiotherapy and agreement with three prediction equations.

BACKGROUND: Bioelectrical impedance spectroscopy (BIS) may be more accurate in determining total body water (TBW) than bioelectrical impedance analysis (BIA). The present study compared the agreement between three TBW prediction equations developed using BIA and BIS-derived TBW in oncology outpatients. METHODS: A cross-sectional, observational study was conducted in 37 outpatients receiving radiotherapy (27 males/10 females, aged 68.3 +/- 10.2 years). TBW was estimated by BIS (TBW(BIS)) and three BIA TBW prediction equations (TBW(ca-u): underweight cancer patients; TBW(ca-n): normal-weight cancer patients; and TBW(rad): patients receiving radiotherapy). Bland-Altman analyses determined agreement between methods. BIS-derived TBW using new resistivity constants was calculated. RESULTS: The mean +/- SD of TBW estimated by BIS was 39.8 +/- 8.3 L, which was significantly different from the prediction equations; TBW(rad) 35.1 +/- 7.9 L, TBW(ca-u) 33.1 +/- 7.5 L and TBW(ca-n) 32.3 +/- 7.3 L, (P < 0.001). Using new resistivity constants, TBW was 36.2 +/- 8.1 L but this still differed from the equations (P < 0.001). Bias between TBW(BIS) and that predicted by the equations was in the range 4.7-7.4 L or 1.1-3.9 L using new resistivity constants. CONCLUSIONS: TBW estimated by BIS cannot be directly compared with oncology-specific BIA equations, suggesting that BIS cannot be used at the group level in outpatients receiving radiotherapy. There was a reduced bias with BIS using new resistivity constants; however, further research should determine any advantage of BIS over BIA in this population.

Joint 24th Medicines for Europe and 21st International Generic and Biosimilars Association (IGBA) Annual Conference (June 13-15, 2018 - Budapest, Hungary).

The joint 24th Medicines for Europe and 21st International Generic and Biosimilars Association (IGBA) Annual Conference brought together key industry leaders and more than 200 participants in Budapest, Hungary, to discuss both challenges and opportunities for the global generic, biosimilar and value added medicines industries. Challenges relating to sustainability were debated with key experts, who shared perspectives on topics such as medicines shortages, use of data, Brexit, international regulatory cooperation, the E.U. Falsified Medicines Directive (FMD) and the potential impact of antimicrobial resistance. European industry leaders outlined the period of intense preparations needed to ensure compliance by the early 2019 milestones for the FMD and Brexit. The conference also anticipated exciting opportunities for the industry and broadly welcomed the European Commission's legislative proposal for a (long-awaited and much discussed) E.U. Supplementary Protection Certificate manufacturing waiver. Medicines for Europe and IGBA are committed to supporting and strengthening such policy initiatives aimed at boosting European competitiveness, increasing investments in R+D for biosimilar medicines, and most importantly, delivering faster access to medicines for patients. The importance of not forgetting that the ultimate aim of the industry was to facilitate patient access to necessary medicines was stressed throughout the conference. As the conference took place in Hungary, László György (State Secretary for Economic Strategy and Regulation at the Ministry of Innovation and Technology) spoke about the role of technology innovation and access to medicines in the country and the role of his ministry. He indicated that the primary goal of the newly established ministry was to maintain and improve decent living conditions in the light of aging populations and enhance the competitiveness of the pharmaceutical industry, "which plays a decisive role in the Hungarian economy."

Immunoregulatory subsets of the T helper and T suppressor cell populations in homosexual men with chronic unexplained lymphadenopathy.

Unexplained, generalized lymphadenopathy in homosexual men, which can be a prodrome to the acquired immunodeficiency syndrome, is associated with impaired cell-mediated immunity, a low ratio of T helper-inducer to T suppressor-cytotoxic cells (defined by the T4 and T8 monoclonal antibodies), and hypergammaglobulinemia. We performed double-marker studies on T cells by using a panel of monoclonal antibodies (Ia, T17, TQ1, and Leu-8), which reportedly detect activation or functional subsets of the T4 and T8 T cell populations. The T4:TQ1- or T4:Leu-8- subset, which is the major helper subset for B cell responses, is normally represented in lymphadenopathy patients. A depression in the reciprocal subset, T4:TQ1+ or T4:Leu-8+, accounts for the T4 T cell defect. Similarly, the TQ1 and Leu-8 markers delineate the abnormality of T8 T cells: the T8:TQ1- or T8:Leu-8- subset is elevated, whereas the T8:TQ1+ or T8:Leu-8+ subset is normally represented. We found no evidence of excessive activation of T4 T cells by using the T17 or Ia monoclonal antibodies. We did find an overall increase in Ia-positive T cells; however, this was due to increased T8:Ia+ cells. In functional studies, immunoglobulin production induced by pokeweed was subnormal. Most lymphadenopathy patients had normal T helper cell function when combined with normal B cells. The dampened pokeweed responses could be partially explained by depression of the T4:TQ1+ (or T4:Leu-8+) subset (which has minor help-associated function) and/or greater than expected suppression. However, subnormal pokeweed responses could not be totally explained by immunoregulatory T cell abnormalities because we also found an intrinsic defect in the B cell responses of lymphadenopathy patients.

DNA rearrangements associated with multiple consecutive directed antigenic switches in Trypanosoma brucei.

Changes in variant surface glycoprotein (Vsg) expression allow Trypanosoma brucei to elude the immune response. The expressed vsg is always located at the telomeric end of a polycistronic transcription unit known as an expression site (ES). Although there are many ESs, only one is active at any particular time. The mechanisms regulating ES transcription and switching are unknown. Chromosome rearrangements within or upstream of the ES have been described to occur in occasional switch events, but no changes have been consistently associated with switching. We inserted the drug resistance genes neo and ble, conferring resistance to G418 and phleomycin, respectively, 1 kb downstream of "silent" ES promoters. This demonstrated that short-range transcription could be achieved from a silent ES promoter. From one initial transformant clone, panels of independent consecutive on-off-on switch clones were generated and analyzed. The first activation of the neo-targeted ES was always associated with deletion of the upstream tandem promoter in this ES, but no further rearrangements were detected in consecutive off-on switches of this ES. On the other hand, direct analysis of ES promoters showed that deletions and duplications occurred elsewhere. Activation of a ble-tagged 300-kb chromosome could not be achieved, but phleomycin-resistant clones could be obtained. One such clone arose from recombination between three ESs. Taken together, our experiments suggest that ES switching may occur after a period of chromosomal interactivity that may or may not leave tangible evidence in the form of detectable sequence changes.

rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.

An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.

Preliminary investigation of urine N-telopeptide concentration as a biomarker of bone resorption in dogs receiving glucocorticoids.

OBJECTIVES: The influence of glucocorticoid therapy on bone resorption in dogs using a urine N-telopeptide assay was investigated. MATERIALS AND METHODS: Thirty-one dogs receiving oral glucocorticoids and 31 age-matched healthy control dogs were enrolled. Urine N-telopeptide concentration was measured using a commercially available immunoassay and results were expressed as a ratio against urinary creatinine concentration. Dogs receiving glucocorticoids were divided into three subgroups based on daily glucocorticoid dose and three subgroups based on treatment duration. Urine N-telopeptide concentration was then compared between groups. RESULTS: Urine N-telopeptide concentration was significantly higher in dogs receiving glucocorticoids compared to the control group. CLINICAL SIGNIFICANCE: This preliminary study demonstrates significant increase in urine N-telopeptide concentration in dogs receiving glucocorticoid therapy compared to control dogs. Further studies are needed to assess whether this increase in urine N-telopeptide concentration correlates with decreases in bone mineral density as has been identified in humans.