Quality of routine health data at the onset of the COVID-19 pandemic in Ethiopia, Haiti, Laos, Nepal, and South Africa.

BACKGROUND: During the COVID-19 pandemic, governments and researchers have used routine health data to estimate potential declines in the delivery and uptake of essential health services. This research relies on the data being high quality and, crucially, on the data quality not changing because of the pandemic. In this paper, we investigated those assumptions and assessed data quality before and during COVID-19. METHODS: We obtained routine health data from the DHIS2 platforms in Ethiopia, Haiti, Lao People's Democratic Republic, Nepal, and South Africa (KwaZulu-Natal province) for a range of 40 indicators on essential health services and institutional deaths. We extracted data over 24 months (January 2019-December 2020) including pre-pandemic data and the first 9 months of the pandemic. We assessed four dimensions of data quality: reporting completeness, presence of outliers, internal consistency, and external consistency. RESULTS: We found high reporting completeness across countries and services and few declines in reporting at the onset of the pandemic. Positive outliers represented fewer than 1% of facility-month observations across services. Assessment of internal consistency across vaccine indicators found similar reporting of vaccines in all countries. Comparing cesarean section rates in the HMIS to those from population-representative surveys, we found high external consistency in all countries analyzed. CONCLUSIONS: While efforts remain to improve the quality of these data, our results show that several indicators in the HMIS can be reliably used to monitor service provision over time in these five countries.

Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164-Cys164 and Cys181-Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181-Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181-Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

Associations between the stringency of COVID-19 containment policies and health service disruptions in 10 countries.

BACKGROUND: Disruptions in essential health services during the COVID-19 pandemic have been reported in several countries. Yet, patterns in health service disruption according to country responses remain unclear. In this paper, we investigate associations between the stringency of COVID-19 containment policies and disruptions in 31 health services in 10 low- middle- and high-income countries in 2020. METHODS: Using routine health information systems and administrative data from 10 countries (Chile, Ethiopia, Ghana, Haiti, Lao People's Democratic Republic, Mexico, Nepal, South Africa, South Korea, and Thailand) we estimated health service disruptions for the period of April to December 2020 by dividing monthly service provision at national levels by the average service provision in the 15 months pre-COVID (January 2019-March 2020). We used the Oxford COVID-19 Government Response Tracker (OxCGRT) index and multi-level linear regression analyses to assess associations between the stringency of restrictions and health service disruptions over nine months. We extended the analysis by examining associations between 11 individual containment or closure policies and health service disruptions. Models were adjusted for COVID caseload, health service category and country GDP and included robust standard errors. FINDINGS: Chronic disease care was among the most affected services. Regression analyses revealed that a 10% increase in the mean stringency index was associated with a 3.3 percentage-point (95% CI -3.9, -2.7) reduction in relative service volumes. Among individual policies, curfews, and the presence of a state of emergency, had the largest coefficients and were associated with 14.1 (95% CI -19.6, 8.7) and 10.7 (95% CI -12.7, -8.7) percentage-point lower relative service volumes, respectively. In contrast, number of COVID-19 cases in 2020 was not associated with health service disruptions in any model. CONCLUSIONS: Although containment policies were crucial in reducing COVID-19 mortality in many contexts, it is important to consider the indirect effects of these restrictions. Strategies to improve the resilience of health systems should be designed to ensure that populations can continue accessing essential health care despite the presence of containment policies during future infectious disease outbreaks.

Tracking health system performance in times of crisis using routine health data: lessons learned from a multicountry consortium.

COVID-19 has prompted the use of readily available administrative data to track health system performance in times of crisis and to monitor disruptions in essential healthcare services. In this commentary we describe our experience working with these data and lessons learned across countries. Since April 2020, the Quality Evidence for Health System Transformation (QuEST) network has used administrative data and routine health information systems (RHIS) to assess health system performance during COVID-19 in Chile, Ethiopia, Ghana, Haiti, Lao People's Democratic Republic, Mexico, Nepal, South Africa, Republic of Korea and Thailand. We compiled a large set of indicators related to common health conditions for the purpose of multicountry comparisons. The study compiled 73 indicators. A total of 43% of the indicators compiled pertained to reproductive, maternal, newborn and child health (RMNCH). Only 12% of the indicators were related to hypertension, diabetes or cancer care. We also found few indicators related to mental health services and outcomes within these data systems. Moreover, 72% of the indicators compiled were related to volume of services delivered, 18% to health outcomes and only 10% to the quality of processes of care. While several datasets were complete or near-complete censuses of all health facilities in the country, others excluded some facility types or population groups. In some countries, RHIS did not capture services delivered through non-visit or nonconventional care during COVID-19, such as telemedicine. We propose the following recommendations to improve the analysis of administrative and RHIS data to track health system performance in times of crisis: ensure the scope of health conditions covered is aligned with the burden of disease, increase the number of indicators related to quality of care and health outcomes; incorporate data on nonconventional care such as telehealth; continue improving data quality and expand reporting from private sector facilities; move towards collecting patient-level data through electronic health records to facilitate quality-of-care assessment and equity analyses; implement more resilient and standardized health information technologies; reduce delays and loosen restrictions for researchers to access the data; complement routine data with patient-reported data; and employ mixed methods to better understand the underlying causes of service disruptions.

Direct evidence of edge-to-face CH/π interaction for PAR-1 thrombin receptor activation.

Heptapeptide SFLLRNP is a receptor-tethered ligand of protease-activated receptor 1 (PAR-1), and its Phe at position 2 is essential for the aggregation of human platelets. To validate the structural elements of the Phe-phenyl group in receptor activation, we have synthesized a complete set of S/Phe/LLRNP peptides comprising different series of fluorophenylalanine isomers (Fn)Phe, where n = 1, 2, 3, and 5. Phe-2-phenyl was strongly suggested to be involved in the edge-to-face CH/π interaction with the receptor aromatic group. In the present study, to prove this receptor interaction definitively, we synthesized another series of peptide analogs containing (F4)Phe-isomers, with the phenyl group of each isomer possessing only one hydrogen atom at the ortho, meta, or para position. When the peptides were assayed for their platelet aggregation activity, S/(2,3,4,6-F4)Phe/LLRNP and S/(2,3,4,5-F4)Phe/LLRNP exhibited noticeable activity (34% and 6% intensities of the native peptide, respectively), whereas S/(2,3,5,6-F4)Phe/LLRNP was completely inactive. The results indicated that, at the ortho and meta positions but not at the para position, benzene-hydrogen atoms are required for the CH/π interaction to activate the receptor. The results provided a decisive evidence of the molecular recognition property of Phe, the phenyl benzene-hydrogen atom of which participates directly in the interaction with the receptor aromatic π plane.

Evaluation of a new point-of-care quantitative reverse transcription polymerase chain test for detecting severe acute respiratory syndrome coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading worldwide, and the resultant disease, coronavirus disease (COVID-19), has become a global pandemic. Although there are multiple methods for detecting SARS-CoV-2, there are some issues with such tests, including long processing time, expense, low sensitivity, complexity, risk of contamination, and user friendly. This study evaluated the reproducibility and usability of a new point-of-care test (POCT) using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for detecting SARS-CoV-2. METHODS: Samples from 96 patients with suspected SARS-CoV-2 infection were assessed using the real-time qRT-PCR-based POCT and the conventional real-time qRT-PCR method based on the Japanese National Institute of Infectious Diseases guidelines (registration number: jRCT1032200025). RESULTS: The real-time qRT-PCR-based POCT had a positive agreement rate of 90.0% (18/20), a negative agreement rate of 100% (76/76), and a total agreement rate of 97.9% (94/96), and the significantly high score of questionnaire survey (total score p < 0.0001). In the two cases in which real-time qRT-PCR-based POCT results did not match conventional real-time qRT-PCR test results, the SARS-CoV-2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real-time qRT-PCR. All patients could be triaged within 1 day using the real-time qRT-PCR-based POCT without invalid reports. CONCLUSIONS: The real-time qRT-PCR-based POCT not only had high reproducibility and useability but also allowed rapid patient triage. Therefore, it may be helpful in clinical settings.

Design of substrates and inhibitors of G protein-coupled receptor kinase 2 (GRK2) based on its phosphorylation reaction.

The G protein-coupled receptor kinase (GRK) family consists of seven cytosolic serine/threonine (Ser/Thr) protein kinases, and among them, GRK2 is involved in the regulation of an enormous range of both G protein-coupled receptors (GPCRs) and non-GPCR substrates that participate in or regulate many critical cellular processes. GRK2 dysfunction is associated with multiple diseases, including cancers, brain diseases, cardiovascular and metabolic diseases, and therefore GRK2-specific substrates/inhibitors are needed not only for studies of GRK2-mediated cellular functions but also for GRK2-targeted drug development. Here, we first review the structure, regulation and functions of GRK2, and its synthetic substrates and inhibitors. We then highlight recent work on synthetic peptide substrates/inhibitors as promising tools for fundamental studies of the physiological functions of GRK2, and as candidates for applications in clinical diagnostics.

A high-affinity peptide substrate for G protein-coupled receptor kinase 2 (GRK2).

We synthesized a previously identified ß-tubulin-derived G protein-coupled receptor kinase 2 (GKR2) peptide (GR-11-1; DEMEFTEAESNMN) and its amino-terminal extension (GR-11-1-N; GEGMDEMEFTEAESNMN) and carboxyl-terminal extension (GR-11-1-C; DEMEFTEAESNMNDLVSEYQ) peptides with the aim of finding a high-affinity peptide substrate for GRK2. GR-11-1-C showed high affinity for GRK2, but very low affinity for GKR5. Its specificity and sensitivity for GKR2 were greater than those of GR-11-1 and GR-11-1-N. These findings should be useful in designing tools for probing GKR2-mediated intracellular signaling pathways, as well as GRK2-specific drugs.

Ph-like acute lymphoblastic leukemia with a novel PAX5-KIDINS220 fusion transcript.

Although "paired box 5" (PAX5)-related fusion genes are well documented in childhood B-cell precursor acute lymphoblastic leukemia (ALL), these types of fusion with the exception of PAX5-JAK2 are rarely seen in patients with gene expression profiles similar to those of BCR-ABL1 (Philadelphia)-positive ALL (Ph-like ALL). We report a novel fusion of the genes PAX5 and "kinase D-interacting substrate of 220 kDa" (KIDINS220, also known as ARMS) in a Ph-like ALL. As PAX5 is a master regulator of B-lymphocyte differentiation, PAX5 rearrangements induce a differentiation block in B lymphocytes. KIDINS220 is a mediator of multiple receptor signaling pathways, interacts with both T- and B-cell receptors, and is necessary for sustained extracellular signal-regulated kinase (ERK) signaling. Although functional studies are needed, the PAX5-KIDINS220 fusion protein might not only inhibit wild-type PAX5 function, but also promote sustained activation of the ERK signaling pathway through upregulation of KIDINS220. © 2016 Wiley Periodicals, Inc.

Reversal of efflux of an anticancer drug in human drug-resistant breast cancer cells by inhibition of protein kinase Cα (PKCα) activity.

P-glycoprotein (Pgp) is a 170-kDa transmembrane protein that mediates the efflux of anticancer drugs from cells. Pgp overexpression has a distinct role in cells exhibiting multidrug resistance (MDR). We examined reversal of drug resistance in human MDR breast cancer cells by inhibition of protein kinase Cα (PKCα) activity, which is associated with Pgp-mediated efflux of anticancer drugs. PKCα activity was confirmed by measurement of phosphorylation levels of a PKCα-specific peptide substrate (FKKQGSFAKKK-NH2), showing relatively higher basal activity in drug-resistant MCF-7/ADR cells (84 %) than that in drug-sensitive MCF-7 cells (63 %). PKCα activity was effectively suppressed by the PKC inhibitor, Ro-31-7549, and reversal of intracellular accumulation of doxorubicin was observed by inhibition of PKCα activity in MCF-7/ADR cells compared with their intrinsic drug resistance. Importantly, increased accumulation of doxorubicin could enhance the therapeutic efficacy of doxorubicin in MDR cells significantly. These results suggest a potential for overcoming MDR via inhibition of PKCα activity with conventional anticancer drugs.

Role of amino acid residues surrounding the phosphorylation site in peptide substrates of G protein-coupled receptor kinase 2 (GRK2).

A series of amino acid substitutions was made in a previously identified ß-tubulin-derived GRK2 substrate peptide (404DEMEFTEAESNMN416) to examine the role of amino acid residues surrounding the phosphorylation site. Anionic amino acid residues surrounding the phosphorylation site played an important role in the affinity for GRK2. Compared to the original peptide, a modified peptide (Ac-EEMEFSEAEANMN-NH2) exhibited markedly higher affinity for GRK2, but very low affinity for GRK5, suggesting that it can be a sensitive and selective peptide for GRK2.

Identification of novel kinase fusion transcripts in paediatric B cell precursor acute lymphoblastic leukaemia with IKZF1 deletion.

Activating tyrosine kinase mutations or cytokine receptor signalling alterations have attracted attention as therapeutic targets for high-risk paediatric acute lymphoblastic leukaemia (ALL). We identified two novel kinase fusions, OFD1-JAK2 and NCOR1-LYN, in paediatric ALL patients with IKZF1 deletion, by mRNA sequencing. The patient with CSF2RA-CRLF2 also harboured IGH-EPOR. All these patients had high-risk features, such as high initial white blood cell counts and initial poor response to prednisolone. The functional analysis of these novel fusions is on-going to determine whether these genetic alterations can be targeted by drugs.

Potential new chemotherapy strategy for human ovarian carcinoma with a novel KSP inhibitor.

Among synthetic kinesin spindle protein (KSP) inhibitor compounds, KPYB10602, a six-member lactam-fused carbazole derivative was the most potent in vitro against cell growth of human ovarian cancer, A2780. KPYB10602 caused dose-dependent suppression of tumor growth in vivo. Mitotic arrest due to KPYB10602 was confirmed in vitro, and was characterized by inhibition of securin degradation. Apoptosis after mitotic arrest was associated with an increase in the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Increase of reactive oxygen species (ROS) and caspase pathway were also involved. Furthermore, KPYB10602 caused little neurotoxicity in vivo. Therefore, KPYB10602 could be a promising candidate as an anti-tumor agent with reduced adverse events for treating human ovarian cancer.

Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is frequently used to monitor phosphorylated peptides or protein kinase activities. However, few reports have compared a radioactivity assay with MALDI-TOF-MS analysis. We analyzed the phosphorylation ratios of 23 peptide substrates for G protein-coupled receptor kinase 2 (GRK2) with different lengths and numbers of negatively charged amino acids by MALDI-TOF-MS. We then examined the correlations between the phosphorylation ratios determined by MALDI-TOF-MS and the radioactivity levels (counts per minute, CPM) determined using a radioactive assay. Using MALDI-TOF-MS, the phosphorylation ratios were greater in the negative mode than in the positive mode. The phosphorylation ratio measured in the negative mode was strongly correlated with the CPM (r = 0.86). The number of acidic amino acids was related to the phosphorylation of peptide substrates by GRK2 (r = 0.53 and 0.46 for the phosphorylation ratio and CPM, respectively). These results suggest that MALDI-TOF-MS is an alternative to radioactive assays for monitoring phosphorylated peptides.

Expression and characterization of myristoylated preS1-conjugated nanocages for targeted cell delivery.

Lipid modification of proteins plays key roles in cellular signaling pathways. We describe the development of myristoylated preS1-nanocages (myr-preS1-nanocages) that specifically target human hepatocyte-like HepaRG cells in which a specific receptor-binding peptide (preS1) is joined to the surface of naturally occurring ferritin cages. Using a genetic engineering approach, the preS1 peptide was joined to the N-terminal regions of the ferritin cage via flexible linker moieties. Myristoylation of the preS1 peptide was achieved by co-expression with yeast N-myristoyltransferase-1 in the presence of myristic acid in Escherichia coli cells. The myristoylated preS1-nanocages exhibited significantly greater specificity for human hepatocyte-like HepaRG cells than the unmyristoylated preS1-nanocages. These results suggest that the lipid-modified nanocages have great potential for effective targeted delivery to specific cells.

An overall characterization of pediatric acute lymphoblastic leukemia with CRLF2 overexpression.

For an overall characterization of pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) with CRLF2 overexpression (OE), we conducted genetic analysis of CRLF2 in 167 pediatric BCPALL patients. CRLF2 OE was detected in 30 (18%) of 167 patients, the P2RY8-CRLF2 fusion was identified in only 3 (1.8%) of 167 patients, all of which demonstrated CRLF2 OE. Moreover, CRLF2 gain was identified in 18 (11%) of 167 patients. Messenger RNA sequencing revealed a novel fusion transcript, CSF2RA-CRLF2, in a case with CRLF2 OE, suggesting that this fusion is associated with CRLF2 OE. In survival analysis, no significant differences in 5-year event-free survival (EFS) and overall survival were observed between patients with and without CRLF2 OE (70.7 vs. 75.4%, log rank P = 0.68 and 96.4 vs. 82.1%, log rank P = 0.11, respectively). However, a significant difference in 5-year EFS between CRLF2 OE patients with and without IKZF1 deletion was observed (44.4 vs. 83.1%, log rank P = 0.02). In multivariate analysis, only IKZF1 deletion was a significant predictor of inferior OS (hazard ratio: 2.427, P = 0.04).These findings suggest that CRLF2 OE is not an independent prognostic factor in pediatric BCPALL.

Dyskeratosis congenita complicated by hepatic fibrosis with hepatic vein thrombosis.

We describe the case of 2 male siblings with dyskeratosis congenita (DC). Extensive genetic analysis failed to identify a causative genetic abnormality. The elder brother developed hepatic fibrosis accompanied with hepatic vein thrombosis at the age of 9 years. Recent studies have found that patients with DC sometimes develop hepatic complications, including cirrhosis. However, little is known about hepatic complications in patients with DC who lack these mutations. Further genetic studies are required to understand the relationship between DC and hepatic complications. In addition, although danazol can sometimes be effective for treating bone marrow failure, hepatotoxicity can be a major complication. Therefore, when danazol is administered to patients with DC, careful monitoring for hepatic complications is important.

Acute lymphoblastic leukemia developing in a patient with Noonan syndrome harboring a PTPN11 germline mutation.

Noonan syndrome (NS) is a congenital genetic disorder characterized by certain facial features, short stature, and congenital heart disease. The disorder is caused by genetic alterations in the RAS/MAPK signal pathway. NS patients show a predisposition to malignancy; however, acute lymphoblastic leukemia (ALL) is rarely reported. Here, we describe a NS patient with B-cell precursor ALL (BCP-ALL) harboring a hyperdiploid karyotype and a PTPN11 germline mutation (c.922A>G; p.N308D). We also discuss the relationship between the hyperdiploid karyotype and genetic alterations in the RAS/MAPK pathway in BCP-ALL.

Reduction of inorganic phosphate-induced human smooth muscle cells calcification by inhibition of protein kinase A and p38 mitogen-activated protein kinase.

High levels of serum phosphate are associated with calcification of human smooth muscle cells (HSMCs). We investigated whether inhibition of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signals [p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)] can reduce inorganic phosphate (Pi)-induced HSMC calcification. Inhibition of PKA or p38 MAPK by inhibitors or small interfering RNAs (siRNAs) reduced Ca levels and alkaline phosphatase activities in HSMCs treated with high Pi, but inhibition of ERK1/2 and JNK showed no significant changes. Moreover, there were no significant changes in cell viability on adding siRNAs and three inhibitors (PKA, p38, and MEK1/2), but JNK inhibitor slightly reduced cell viability. These results show that PKA and p38 MAPK are involved in the Pi-induced calcification of HSMCs, and may be good targets for reducing vascular calcification.