GINSA: an accumulator for paired locality and next-generation small ribosomal subunit sequence data.

MOTIVATION: Motivated by the challenges of decentralized genetic data spread across multiple international organizations, GINSA leverages the Global Biodiversity Information Facility infrastructure to automatically retrieve and link small ribosomal subunit sequences with locality information. RESULTS: Testing on taxa from major organism groups demonstrates broad applicability across taxonomic levels and dataset sizes. AVAILABILITY AND IMPLEMENTATION: GINSA is a freely accessible Python program under the MIT License and can be installed from PyPI via pip.

Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity.

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal. Here, we establish a method for long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore, we achieve single-amino acid level sensitivity, enabling sequencing of combinations of amino acid substitutions across long protein strands. For greater sequencing accuracy, we demonstrate the ability to reread individual protein molecules, spanning hundreds of amino acids in length, multiple times, and explore the potential for high accuracy protein barcode sequencing. Further, we develop a biophysical model that can simulate raw nanopore signals a priori, based on amino acid volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine intact, folded protein domains for complete end-to-end analysis. These results provide proof-of-concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.

Potential application of the haematology analyser XN-31 prototype for field malaria surveillance in Kenya.

BACKGROUND: Simple and accurate diagnosis is a key component of malaria control programmes. Microscopy is the current gold standard, however it requires extensive training and the results largely rely on the skill of the microscopists. Malaria rapid diagnostic tests (RDT) can be performed with minimal training and offer timely diagnosis, but results are not quantitative. Moreover, some Plasmodium falciparum parasites have evolved and can no longer be detected by existing RDT. Developed by the Sysmex Corporation, the XN-31 prototype (XN-31p) is an automated haematology analyser capable of detecting Plasmodium-infected erythrocytes and providing species differentiation and stage specific parasite counts in venous blood samples without any preparation in approximately one minute. However, factors such as stable electricity supply in a temperature-controlled room, cost of the instrument and its initial set-up, and need for proprietary reagents limit the utility of the XN-31p across rural settings. To overcome some of these limitations, a hub and spoke diagnosis model was designed, in which peripheral health facilities were linked to a central hospital where detection of Plasmodium infections by the XN-31p would take place. To explore the feasibility of this concept, the applicability of capillary blood samples with the XN-31p was evaluated with respect to the effect of sample storage time and temperature on the stability of results. METHODS: Paired capillary and venous blood samples were collected from 169 malaria-suspected outpatients in Homa Bay County Referral Hospital, Kenya. Malaria infections were diagnosed with the XN-31p, microscopy, RDT, and PCR. Capillary blood samples were remeasured on the XN-31p after 24 h of storage at either room (15-25 °C) or chilled temperatures (2-8 °C). RESULTS: Identical results in malaria diagnosis were observed between venous and capillary blood samples processed immediately after collection with the XN-31p. Relative to PCR, the sensitivity and specificity of the XN-31p with capillary blood samples were 0.857 and 1.000, respectively. Short-term storage of capillary blood samples at chilled temperatures had no adverse impact on parasitaemia and complete blood counts (CBC) measured by the XN-31p. CONCLUSION: These results demonstrate the potential of the XN-31p to improve routine malaria diagnosis across remote settings using a hub and spoke model.

Determination of a T cell receptor of potent CD8+ T cells against simian immunodeficiency virus infection in Burmese rhesus macaques.

Cumulative studies on human immunodeficiency virus (HIV)-infected individuals have shown association of major histocompatibility complex class I (MHC-I) polymorphisms with lower viral load and delayed AIDS progression, suggesting that HIV replication can be controlled by potent CD8+ T-cell responses. We have previously established an AIDS model of simian immunodeficiency virus (SIV) infection in Burmese rhesus macaques and found a potent CD8+ T cell targeting the Mamu-A1*065:01-restricted Gag241-249 epitope, which is located in a region corresponding to the HIV Gag240-249 TW10 epitope restricted by a protective MHC-I allele, HLA-B*57. In the present study, we determined a T cell receptor (TCR) of this Gag241-249 epitope-specific CD8+ T cell. cDNA clones encoding TCR-α and TCR-ß chains were obtained from a Gag241-249-specific CD8+ T-cell clone. Coexpression of these TCR-α and TCR-ß cDNAs resulted in reconstitution of a functional TCR specifically detected by Gag241-249 epitope-Mamu-A1*065:01 tetramer. Two of three previously-reported CD8+ T-cell escape mutations reduced binding affinity of Gag241-249 peptide to Mamu-A1*065:01 but the remaining one not. This is consistent with the data obtained by molecular modeling of the epitope-MHC-I complex and TCR. These results would contribute to understanding how viral CD8+ T-cell escape mutations are selected under structural constraint of viral proteins.

Immunogenicity of repeated Sendai viral vector vaccination in macaques.

Induction of durable cellular immune responses by vaccination is an important strategy for the control of persistent pathogen infection. Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. Repeated vaccination may contribute to durable memory T-cell induction, but anti-vector antibodies could be an obstacle to its efficacy. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient T-cell induction in macaques. Here, we examined whether repeated SeV vector vaccination with short intervals can enhance antigen-specific CD8(+) T-cell responses. Four rhesus macaques possessing the MHC-I haplotype 90-120-Ia were immunized three times with intervals of three weeks. For the vaccination, we used replication-defective F-deleted SeV vectors inducing CD8(+) T-cell responses specific for simian immunodeficiency virus Gag(206-216) and Gag(241-249), which are dominant epitopes restricted by 90-120-Ia-derived MHC-I molecules. All four animals showed higher Gag(206-216)-specific and Gag(241-249)-specific CD8(+) T-cell responses after the third vaccination than those after the first vaccination, indicating enhancement of antigen-specific CD8(+) T-cell responses by the second/third SeV vector vaccination even with short intervals. These results suggest that repeated SeV vector vaccination can contribute to induction of efficient and durable T-cell responses.

Intranasal Sendai viral vector vaccination is more immunogenic than intramuscular under pre-existing anti-vector antibodies.

Viral vectors are promising vaccine tools for eliciting potent cellular immune responses. Pre-existing anti-vector antibodies, however, can be an obstacle to their clinical use in humans. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient CD8(+) T-cell induction in macaques. Here, we investigated the immunogenicity of SeV vector vaccination in the presence of anti-SeV antibodies. We compared antigen-specific CD8(+) T-cell responses after intranasal or intramuscular immunization with a lower dose (one-tenth of that in our previous studies) of SeV vector expressing simian immunodeficiency virus Gag antigen (SeV-Gag) between naive and pre-SeV-infected cynomolgus macaques. Intranasal SeV-Gag immunization efficiently elicited Gag-specific CD8(+) T-cell responses not only in naive but also in pre-SeV-infected animals. In contrast, intramuscular SeV-Gag immunization induced Gag-specific CD8(+) T-cell responses efficiently in naive but not in pre-SeV-infected animals. These results indicate that both intranasal and intramuscular SeV administrations are equivalently immunogenic in the absence of anti-SeV antibodies, whereas intranasal SeV vaccination is more immunogenic than intramuscular in the presence of anti-SeV antibodies. It is inferred from a recent report investigating the prevalence of anti-SeV antibodies in humans that SeV-specific neutralizing titers in more than 70% of people are no more than those at the SeV-Gag vaccination in pre-SeV-infected macaques in the present study. Taken together, this study implies the potential of intranasal SeV vector vaccination to induce CD8(+) T-cell responses even in humans, suggesting a rationale for proceeding to a vaccine clinical trial using this vector.

Cut-off value for normal versus abnormal right-to-left shunt percentages using (99m)Tc-macroaggregated albumin.

OBJECTIVE: To determine the cut-off value for distinguishing a normal versus an abnormal right-to-left shunt percentage on lung perfusion scintigraphy using (99m)Tc-macroaggregated albumin (MAA). MATERIALS AND METHODS: Fifty-three patients (eight patients with a right-to-left shunt and 45 without a right-to-left shunt) who underwent MAA whole-body imaging for the evaluation of right-to-left shunts were divided into group 1 (eight patients with brain MAA uptake) and group 2 (45 patients without brain MAA uptake). Moreover, group 2 was subdivided into two categories (groups 2a and 2b) based on the results of lung computed tomography, electrocardiography examinations, and pulmonary function tests. The average and standard deviation (SD) of each group were compared. In addition, we estimated the cut-off value for a normal right-to-left shunt percentage using whole-body imaging. RESULTS: The average right-to-left shunt percentage values and SD were 23.67±12.17% in group 1, 6.68±1.04% in group 2a, and 6.60±0.84% in group 2b. The shunt percentages of groups 2a and 2b were not significantly different (P=0.77). The estimated normal value (mean±2 SD) of group 2 was 6.64±0.94%. Meanwhile, the cut-off value was estimated as 10% based on the distributions of MAA shunt percentages for groups 1 and 2. CONCLUSION: The normal range (mean±2 SD) was 6.64±1.88%. The cut-off value for the normal right-to-left shunt percentage in MAA scintigraphy was 10%.

Whole-body FDG-PET/CT on rheumatoid arthritis of large joints.

OBJECTIVE: Fluorodeoxyglucose (FDG) uptake in joint lesions in patients with rheumatoid arthritis (RA) reportedly represents the degree of synovial inflammation. Most previous studies have focused on small joints, and the application of whole-body positron emission tomography (PET) combined with computed tomography (CT) (PET/CT) for the evaluation of inflammatory activity in large joints has not been well studied. METHODS: Eighteen patients with RA underwent FDG-PET/CT. FDG uptake in the knee, hip, carpal, wrist, elbow, shoulder, and atlanto-axial joint (total of 13 joints) and in the axillary lymph nodes was evaluated by calculating the maximum standardized uptake value (SUV(max)) and the visual uptake scores as follows: 0, no uptake; 1, slight uptake; 2, moderate uptake (same as in liver); 3, higher than in liver; 4, highest uptake. The number of painful/swollen joints, the white blood cell (WBC) count, and the C-reactive protein (CRP) level were also evaluated. RESULTS: Whole-body FDG-PET/CT delineated large-joint lesions in patients with RA, and the metabolic activity of inflammation was accurately overlaid on the joint anatomy. The total FDG score for all 13 joints was significantly correlated with the CRP level (r = 0.653, p < 0.01, n = 18). The total SUV(max) and the CRP level were weakly, but not significantly, correlated (r = 0.377, p > 0.05). The WBC count was not correlated with any other parameter. The mean number of joints per patient with an FDG uptake score of 2 or more was significantly larger than the mean number of painful/swollen joints (6.2 +/- 3.3 vs. 3.1 +/- 2.7, n = 18, p < 0.01) and both parameters were strongly correlated (r = 0.588, p < 0.01, n = 18). Also, FDG uptake score and SUV of painful/swollen joints were significantly higher than these of not painful/swollen joints. FDG uptake was significantly different from patients of remission and patients of active arthritis. Uptake in the atlanto-axial joint was observed in five (mostly asymptomatic) patients (5/18, 28%), and the uptake score was significantly correlated with the total FDG score (r = 0.669, p < 0.01, n = 18). The axillary lymph nodes score was correlated with the arm joints score. CONCLUSION: FDG-PET/CT represents the inflammatory activity in large joints in patients with RA accurately and sensitively and may be helpful for early evaluations of the extent of RA throughout the whole body including high risk lesion of atlanto-axial joint. Furthermore, the visual FDG uptake score may be useful for evaluating arthritis in large joints.

Gag-specific cytotoxic T-lymphocyte-based control of primary simian immunodeficiency virus replication in a vaccine trial.

Gag-specific cytotoxic T lymphocytes (CTLs) exert strong suppressive pressure on human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. However, it has remained unclear whether they can actually contain primary viral replication. Recent trials of prophylactic vaccines inducing virus-specific T-cell responses have indicated their potential to confer resistance against primary SIV replication in rhesus macaques, while the immunological determinant for this vaccine-based viral control has not been elucidated thus far. Here we present evidence implicating Gag-specific CTLs as responsible for the vaccine-based primary SIV control. Prophylactic vaccination using a Gag-expressing Sendai virus vector resulted in containment of SIVmac239 challenge in all rhesus macaques possessing the major histocompatibility complex (MHC) haplotype 90-120-Ia. In contrast, 90-120-Ia-positive vaccinees failed to contain SIVs carrying multiple gag CTL escape mutations that had been selected, at the cost of viral fitness, in SIVmac239-infected 90-120-Ia-positive macaques. These results show that Gag-specific CTL responses do play a crucial role in the control of wild-type SIVmac239 replication in vaccinees. This study implies the possibility of Gag-specific CTL-based primary HIV containment by prophylactic vaccination, although it also suggests that CTL-based AIDS vaccine efficacy may be abrogated in viral transmission between MHC-matched individuals.