In vivo clonal tracking reveals evidence of haemangioblast and haematomesoblast contribution to yolk sac haematopoiesis.

During embryogenesis, haematopoietic and endothelial lineages emerge closely in time and space. It is thought that the first blood and endothelium derive from a common clonal ancestor, the haemangioblast. However, investigation of candidate haemangioblasts in vitro revealed the capacity for mesenchymal differentiation, a feature more compatible with an earlier mesodermal precursor. To date, no evidence for an in vivo haemangioblast has been discovered. Using single cell RNA-Sequencing and in vivo cellular barcoding, we have unravelled the ancestral relationships that give rise to the haematopoietic lineages of the yolk sac, the endothelium, and the mesenchyme. We show that the mesodermal derivatives of the yolk sac are produced by three distinct precursors with dual-lineage outcomes: the haemangioblast, the mesenchymoangioblast, and a previously undescribed cell type: the haematomesoblast. Between E5.5 and E7.5, this trio of precursors seeds haematopoietic, endothelial, and mesenchymal trajectories.

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity.

The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface-displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthrough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N- and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a "drop-in" version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems-and protein translation apparatuses-to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.

Medical immunity, international law and just war theory.

Under customary international law, the First Geneva Convention and Additional Protocol I, medical personnel are protected against intentional attack. In § 1 of this paper, we survey these legal norms and situate them within the broader international humanitarian law framework. In § 2, we explore the historical and philosophical basis of medical immunity, both of which have been underexplored in the academic literature. In § 3, we analyse these norms as applied to an attack in Afghanistan (2015) by the United States; the United States was attempting to target a Taliban command-and-control centre but inadvertently destroyed a Médecins Sans Frontières hospital instead, killing 42 people. In § 4, we consider forfeiture of medical immunity and, more sceptically, whether supreme emergency could justify infringement of non-forfeited protected status.

Breast cancer gene therapy using an adenovirus encoding human IL-2 under control of mammaglobin promoter/enhancer sequences.

Interleukin-2 (IL-2) has been used clinically for the treatment of some malignancies, but the toxicities associated with systemic IL-2 therapy are a major challenge. Here we have determined whether transcriptional targeting of IL-2 to breast cancer (BrCa) using an engineered human mammaglobin promoter/enhancer (MPE2) is a feasible option for reducing IL-2-associated toxicities while still achieving a meaningful antitumor effect. We have constructed nonreplicating adenovirus vectors encoding either a reporter gene (luciferase) or human IL-2 (hIL-2) complementary DNA under control of the MPE2 sequence, the murine cytomegalovirus immediate early (MCMV) promoter or the human telomerase reverse transcriptase (hTERT) promoter. Luciferase and hIL-2 complementary DNAs under the control of the MPE2 sequence in adenovirus vectors were expressed at high levels in BrCa cells and at lower levels in normal cells of human and murine origin. Cancer specificity of the hTERT promoter was found to be similar to that of the MPE2 promoter in cells of human origin, but reduced specificity in murine cells. The MPE2 regulatory sequence demonstrated excellent tissue specificity in a mouse tumor model. Whereas the MCMV promoter-controlled IL-2 vector generated high liver toxicity in mice, the MPE2-controlled IL-2 vector generated little or no liver toxicity. Both IL-2 vectors exerted significant tumor growth delay; however, attempts to further enhance antitumor activity of the IL-2 vectors by combining with the proapoptotic drug procaspase activating compound 1 (PAC1) were unsuccessful.

An unusual otoscopic finding associated with a type II first branchial cleft anomaly.

OBJECTIVE: We report an interesting case involving a child with a branchial cleft anomaly with two fistulous tracts, one of which was associated with an unusual otoscopic finding. CASE REPORT: A seven-year-old girl presented with an apparent type II first branchial cleft cyst after an acute infection. Parotidectomy and excision of the tract were performed, with subsequent development of pre-auricular swelling three months later. Further surgery was performed to remove a second duplication anomaly of the external auditory canal. Otomicroscopy showed a fibrous band arising from the wall of the canal and attached to the tympanic membrane at the umbo. CONCLUSION: Otoscopic findings on physical examination can be important diagnostic clues in the early recognition of branchial cleft anomalies. The classification system proposed by Work may fail to describe some branchial cleft lesions.

MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and follicular exclusion of anti-double-stranded DNA B cells.

A hallmark of systemic lupus erythematosus and the MRL murine model for lupus is the presence of anti-double-stranded (ds)DNA antibodies (Abs). To identify the steps leading to the production of these Abs in autoimmune mice, we have compared the phenotype and localization of anti-dsDNA B cells in autoimmune (MRL+/+ and lpr/lpr) mice with that in nonautoimmune (BALB/c) mice. Anti-dsDNA B cells are actively regulated in BALB/c mice as indicated by their developmental arrest and accumulation at the T-B interface of the splenic follicle. In the MRL genetic background, anti-dsDNA B cells are no longer developmentally arrested, suggesting an intrinsic B cell defect conferred by MRL background genes. With intact Fas, they continue to exhibit follicular exclusion; however, in the presence of the lpr/lpr mutation, anti-dsDNA B cells are now present in the follicle. Coincident with the altered localization of anti-dsDNA B cells is a follicular infiltration of CD4 T cells. Together, these data suggest that MRL mice are defective in maintaining the developmental arrest of autoreactive B cells and indicate a role for Fas in restricting entry into the follicle.

Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process.

Anti-single stranded DNA (ssDNA) and anti-double stranded DNA (dsDNA) B cells are regulated in non-autoimmune mice. In this report we show that while both anti-ssDNA and anti-dsDNA B cells are blocked in their ability to differentiate into antibody-secreting cells, other phenotypic and functional characteristics distinguish them from one another. Splenic anti-ssDNA B cells are found distributed throughout the B cell follicle, and are phenotypically mature and long-lived. On the other hand, splenic anti-dsDNA B cells are short-lived, exhibit an immature and antigen-experienced phenotype, and localize to the T-B interface of the splenic follicle. Functionally, anti-ssDNA B cells proliferate, albeit suboptimally, in response to anti-IgM, lipopolysaccharide (LPS) and CD40L/IL-4 + anti-IgM stimulation, and tyrosine phosphorylate intracellular proteins upon mIgM cross-linking. Anti-dsDNA B cells, on the other hand, are functionally unresponsive to anti-IgM and LPS stimulation, and do not phosphorylate intracellular proteins, including Syk, upon mIg stimulation. Importantly, anti-DNA B cell anergy is maintained in the absence of T cells since both anti-ssDNA and anti-dsDNA B cells are as efficiently regulated in RAG2(-/-) mice as in their RAG2(+/+) counterparts. Interestingly, the severely anergic state of anti-dsDNA B cells is partially reversible upon stimulation with CD40 ligand and IL-4. In response to these signals, anti-dsDNA B cells remain viable, up-regulate cell surface expression of B7-2 and IgM, and restore their ability to proliferate and phosphorylate Syk upon mIg cross-linking. Collectively, these data suggest that anti-DNA B cell anergy encompasses distinct phenotypes which, even in its most severe form, may be reversible upon stimulation with T cell-derived factors.

Protease inhibitors as antiviral agents.

Currently, there are a number of approved antiviral agents for use in the treatment of viral infections. However, many instances exist in which the use of a second antiviral agent would be beneficial because it would allow the option of either an alternative or a combination therapeutic approach. Accordingly, virus-encoded proteases have emerged as new targets for antiviral intervention. Molecular studies have indicated that viral proteases play a critical role in the life cycle of many viruses by effecting the cleavage of high-molecular-weight viral polyprotein precursors to yield functional products or by catalyzing the processing of the structural proteins necessary for assembly and morphogenesis of virus particles. This review summarizes some of the important general features of virus-encoded proteases and highlights new advances and/or specific challenges that are associated with the research and development of viral protease inhibitors. Specifically, the viral proteases encoded by the herpesvirus, retrovirus, hepatitis C virus, and human rhinovirus families are discussed.

Estimate of the frequency of human immunodeficiency virus type 1 protease inhibitor resistance within unselected virus populations in vitro.

The frequency of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants in virus populations not previously exposed to drug was determined in vitro by using HIV-1RF and the protease inhibitor SC-55389A. Two variants with single mutations responsible for drug resistance (V82A and N88S) were quantifiably isolated after only one round of replication, yielding a crude frequency estimate of at least 1 SC-55389A-resistant variant per 3.5 x 10(5) wild-type infectious units.

Safety and efficacy of transurethral needle ablation of the prostate for symptomatic outlet obstruction.

OBJECTIVES: To examine, in an observational study, the safety and efficacy of transurethral needle ablation (TUNA) of the prostate as a treatment for symptomatic benign prostatic enlargement. PATIENTS AND METHODS: This prospective study included 71 symptomatic men with unequivocal obstruction on pressure-flow urodynamics. The variables measured at baseline and up to 12 months after treatment included the American Urological Association (AUA)-7 symptom index and an added quality-of-life question, the AUA BPH-Impact Index, a sexual function score, transrectal ultrasonography of the prostate, a frequency-volume chart, free-flow uroflowmetry, post-void residual urine volume (PVR) and pressure-flow urodynamics. Transurethral resection of the prostate (TURP) was offered if the symptoms failed to resolve at any time during the follow-up period. TUNA was performed under local anaesthetic and sedation in 63 (89%) men and as a day-case procedure in 10 (14%). Five patients were on warfarin which was not discontinued. RESULTS: There were no serious treatment-related adverse events. Eight of the initial nine patients who were not routinely catheterized after treatment with TUNA developed acute urinary retention. Although some haematuria occurred in all patients, only one (1.4%) developed catheter blockage by clot. There were no problems with bleeding in those patients on warfarin at the time of treatment. The mean (95% confidence interval, CI) AUA-7 index fell from 23 (1.7) to 10.6 (1.8) (P < 0.001, Mann-Whitney U-test) at 12 months, 29 men (41%) had an AUA-7 index of < or = 7. The maximum (95% CI) urinary flow rate increased from 9.0 (0.8) to 11.3 (1.1) mL/s (P < 0.001) and this was accompanied by a small but significant reduction in PVR of 70 (14) mL to 35 (8) mL (P < 0.001 Mann-Whitney U-test). There was a significant reduction in both maximal voiding pressure and detrusor pressure at peak flow at 3 months (Mann-Whitney U-test, both P < 0.001) and at 12 months (P < 0.001, Wilcoxon matched-pairs signed-ranks test). However, 78% of the 45 men undergoing repeat pressure-flow studies at 12 months were unequivocally obstructed according to the Abrams-Griffiths nomogram. The mean (95% CI) prostatic volume fell from 49.0 (4.8) mL at baseline to 40.8 (4.9) mL at 3 months, but this change was not statistically significant (P = 0.011, Mann-Whitney U-test). Two men reported erectile dysfunction, one experienced ejaculatory problems and seven reported an improvement in erectile function after TUNA. During the study, 22 men (31%) underwent TURP. CONCLUSIONS: TUNA is a safe treatment which can be performed as an out-patient procedure under local anaesthesia and sedation in the vast majority of patients. There was no evidence of serious adverse events and no significant adverse effect on sexual function. The symptomatic improvement was sustained at 12 months in most (54%) patients, with modest improvements in peak flow rate, PVR and voiding pressures, indicating that TUNA may result in prolonged symptomatic improvement in a proportion of patients suffering from bladder outlet obstruction. A randomized controlled study against established therapies is now essential to clearly delineate its place in the management of such patients.

A mutation in human immunodeficiency virus type 1 protease at position 88, located outside the active site, confers resistance to the hydroxyethylurea inhibitor SC-55389A.

The hydroxyethylurea human immunodeficiency virus type 1 (HIV-1) protease inhibitors SC-55389A and SC-52151 were used to select drug-resistant variants in vitro. One clinical HIV-1 strain (89-959) and one laboratory HIV-1 strain (LAI) were passaged in peripheral blood mononuclear cells or CEMT4 cells in the presence of SC-55389A. Resistant isolates from both strains consistently had a mutation to serine for asparagine at amino acid 88 (N88S) in the protease gene either alone or in combination with a change to phenylalanine at position 10. The N88S mutation, recreated by oligonucleotide-mediated site-directed mutagenesis in HXB2, was sufficient to confer resistance to SC-55389A. In contrast, SC-52151-resistant variants selected from the monocytotropic strain SF162 had multiple substitutions in the protease gene (I11V, M461, F53L, A71V, and N88D), and the N88D mutation, re-created by oligonucleotide-mediated site-directed mutagenesis in HXB2, did not confer resistance to SC-52151. The potencies of L735,524 and Ro31-8959 were not reduced when these compounds were assayed against variants with either the N88S or N88D substitution. Position 88 is in a helix that lies behind the substrate binding pocket and may indirectly influence inhibitor binding through interactions with the amino acid at position 31. The selected mutations were persistent in the viral populations after more than 20 passages in the absence of drugs. Passaging of virus first in SC-55389A alone and then in combination with SC-52151 resulted in the accumulation of more mutations in the protease gene (L10F, D35E, D37M, I47V, 154L, A71V, V82I, and S88D) and in the selection of a variant that was cross-resistant to multiple protease inhibitors. These results indicate that a mutation in the HIV-1 protease at a position that is located outside of the substrate binding pocket confers resistance to a protease inhibitor and that mutations in the protease gene accumulate with increasing selection pressure and can persist in the absence of selection pressure.

A prospective audit of the use of a prostate clinic.

OBJECTIVE: To assess the efficiency of a prostate clinic and to determine the treatment outcomes and the proportion of patients who could potentially be managed by their General Practitioners (GPs). PATIENTS AND METHODS: Referral letters from GPs were screened by the consultant urologists and appropriate patients seen in the next available prostate clinic. The initial assessment consisted of an International Prostate Symptom Score and a medical history, uroflowmetry, ultrasonographically determined post-void urine volumes, renal function tests and measurement of prostate specific antigen, in addition to a physical examination and a digital rectal examination. Further investigations were requested as required. RESULTS: Over a period of 18 months, 403 patients were seen, 90% of them within 12 weeks from the time of referral. Uroflowmetry was performed in 96% of patients and further urodynamics in 22%. Bladder outlet obstruction was diagnosed in 246 (61%) patients and primary detrusor instability was detected in 20 (5%) patients. Fourteen per cent of patients were returned to the care of the GP following their first visit. The audit identified a potential group of patients (52%) who could be managed by their GP. Seven per cent underwent prostate surgery for the relief of bladder outlet obstruction. CONCLUSION: The prostate clinic significantly reduced the delay for patients to be seen at the hospital and facilitated rapid assessment and investigation, much of which was carried out by a nurse practitioner during the first visit (in most cases). Several patients were identified who could be managed in the community.

Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells.

OBJECTIVES: Nitric oxide synthases (NOS) generate nitric oxide (NO), a second messenger with key regulatory roles. In the cardiovascular system, deficient endothelial NO production is an early, persistent feature of atherosclerosis and vascular injury. Accordingly, the NOS isoforms represent attractive targets for vascular gene therapy. We aimed to generate and evaluate an adenoviral vector for gene transfer of an NOS isoform to vascular cells. METHODS: We constructed a recombinant adenovirus, Ad.nNOS, for gene transfer of the neuronal isoform of NOS (nNOS) and characterized its expression in 293 cells, human vascular smooth muscle cells (hVSMC) and human umbilical vein endothelial cells (HUVEC). NOS expression was analyzed by Western immunoblotting, and NOS enzyme activity in response to receptor-dependent and receptor-independent agonists was determined by Griess assay or by NO chemiluminescence. RESULTS: Ad.nNOS-infected 293 cells expressed high levels of functional nNOS enzyme, even higher than in 293.NOS cells (a cell line that expresses supraphysiologic levels of nNOS). In hVSMC, nNOS activity reached levels 50% of those seen in 293.NOS cells. nNOS expression and activity in hVSMC increased linearly with titer of Ad.nNOS. NO production in hVSMC was stimulated both by calcium ionophore and by physiologic agonists such as acetylcholine or bradykinin. In HUVEC, endogenous NOS activity was significantly augmented by Ad.nNOS infection. Supplementation with the tetrahydrobiopterin precursor sepiapterin enhanced NOS activity in all cells. CONCLUSIONS: Ad.nNOS, a novel adenoviral vector for gene transfer of NOS, generates high-level nNOS expression in a variety of vascular cell types. nNOS activity in hVSMC is physiologically regulated and of a magnitude comparable to native eNOS activity in HUVEC. Our findings demonstrate Ad.nNOS to be a versatile and efficient tool for nNOS gene transfer, with widespread potential applications in cell culture and for gene therapy.

Genetic heterogeneity of the V3 region of the HIV-1 envelope glycoprotein in Brazil. Brazilian Collaborative AIDS Research Group.

OBJECTIVE: To examine the genetic heterogeneity of the V3 region of HIV-1 gp120 from 22 Brazilian HIV-1 specimens. DESIGN: Genetic heterogeneity was examined by DNA sequencing of the C2 V3 region of the HIV-1 envelope (env) gene from polymerase chain reaction (PCR)-amplified HIV-1 DNA. Deduced amino-acid sequences were compared to determine the extent of amino-acid conservation among the Brazilian specimens. Genetic similarity among and between the Brazilian specimens and other previously published HIV-1 isolates was analyzed by principal co-ordinate and DNA parsimony methods. METHODS: A 282 base pair (bp) region of a 1.5 kilo (k) bp PCR-amplified HIV-1 env fragment was sequenced by a Taq dye-labeled primer cycle sequencing reaction. Nucleotide sequences were used to analyze inter-specimen relationships based on overall nucleotide sequence similarity and DNA parsimony principles. RESULTS: Amino-acid comparison showed that 15 of the 35 (43%) residues of the V3 loop were conserved among the Brazilian specimens. Nine of the 22 (40%) Brazilian specimens contained the North American-European GPGR tetrapeptide motif, while eight (36%) contained the GWGR motif, previously reported in Japanese isolates. Principal co-ordinate analysis demonstrated that 19 of the 20 examined Brazilian HIV-1 specimens were more similar to North American and Haitian isolates than to African isolates. Similar results were also obtained by DNA parsimony analysis. CONCLUSION: The majority of the Brazilian specimens examined are more genetically related to North American and Haitian HIV-1 isolates than to African isolates. This finding and the presence of a GWGR V3 loop motif in some Brazilian isolates may be important for vaccine development.

Genetic diversity of human immunodeficiency virus type 1 strains in Kinshasa, Zaire.

The envelope (env) gene of human immunodeficiency virus type 1 (HIV-1) was amplified by polymerase chain reaction (PCR) from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1-infected women from Kinshasa, Zaire. Amplified DNA was directly sequenced with a primer specific for the HIV-1 env C2 region. The predicted amino acid sequences for the C2-V3 region for the 14 specimens are presented. The tetrapeptide sequence, GPGQ, located at the crown of the V3 loop, is conserved in all specimens. The same tetrapeptide sequence is present in the Zairian isolate MAL, but not in other published Zairian isolates (Z6, ELI, Z321, JY1, and NDK). Sequence comparison of the env C2-V3 region among the 14 specimens from Kinshasa revealed a 9-25% range of nucleotide divergence, with an average of 16%. Divergence between the 14 specimens and the Zairian isolates MAL, Z6, ELI, Z321, JY1, and NDK ranged from 13 to 31%. A range of 18-28% nucleotide sequence divergence was demonstrated between the 14 Kinshasa specimens and the North American isolate MN. These results demonstrate the importance of examining HIV-1 samples from diverse geographic origins in the development of effective HIV-1 vaccines.

V3 loops of HIV-1 specimens from pregnant women in Malawi uniformly lack a potential N-linked glycosylation site.

PIP: The HIV-1 env gene was amplified from the peripheral blood mononuclear cells of 14 infected pregnant women in Malawi. Nested polymerase chain reaction (PCR) and DNA sequencing were performed. The PCR product was purified and the C2-V3 region sequenced. Using the similarity function of the multiple aligned sequence editor, 13 of the nucleotide sequences were compared. The interperson variation, based on single base substitutions, ranged from 7.3 to 22.2% (mean 13.6%). All of the sequences showed the tetrapeptide motif at the crown of the V3 loop which is commonly seen among HIV-1 subtypes A, C, D, and E. The C2-V3 coding sequences clustered with the subtype C sequence reported from South Africa. In addition, all of these sequences lacked a potential N-linked glycosylation site found in all HIV-1 sequences except subtype C. In these specimens, the predominant replacement was valine. The role of this site in HIV-1 transmission is controversial.^ieng

Conjugated and cross-conjugated mesomeric betaines. Correlation of electroreduction with structure and physiological activity.

Electroreduction studies were performed on several cross-conjugated mesomeric betaines containing the fused pyrazolium (2) and fused imidazolium (3) ring systems. Studies at acidic pH were of principal interest. Substituent effects for 2 were in line with prior findings, and reduction potentials were comparatively negative (-0.96 to -1.34 V). Reduction potentials fit the modified Hammett equation. Compound 3 was more readily reduced (-0.88 V). The related psi-oxatriazoles (6) gave values in the range of -0.85 to -1.22 V. The electrochemical characteristics are compared with those of the mesoionic sydnones (4) and sydnoneimines (5). These mesoionic compounds were generally reduced at more positive potentials than 2 and 3. A relationship between electroreduction and physiological activity is proposed. The overall results are in keeping with the hypothesis of widespread participation of iminium-type species in biological systems.