The novel Plasmodium berghei protein S14 is essential for sporozoite gliding motility and infectivity.

Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tail-interacting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasion-deficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission.

A Plasmodium apicoplast-targeted unique exonuclease/FEN exhibits interspecies functional differences attributable to an insertion that alters DNA-binding.

The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance.

Stearoyl-CoA desaturase regulates organelle biogenesis and hepatic merozoite formation in Plasmodium berghei.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.

Effects of concentration of hydrophobic component and swelling in saline solutions on mechanical properties of a stretchable hydrogel.

An elastic biopolymer, resilin possesses exceptional qualities such as high stretchability and resilience. Such attributes are utilized in nature by many species for mechanical energy storage to facilitate movement. The properties of resilin are attributed to the balanced combination of hydrophilic and hydrophobic segments. To mimic the properties of resilin, we developed a hydrogel system composed of hydrophilic acrylic acid (AAc) and methacrylamide (MAM) chains and hydrophobic poly(propylene glycol diacrylate) (PPGDA) chains. The gel was produced through free-radical polymerization in 0.8 M NaCl solutions using KPS as an initiator. In these gels, AAc and MAM can form hydrogen bonds, whereas the association between PPGDA chains can lead to hydrophobic domains. The PPGDA concentration affects the level of hydrogen bonding and gel mechanical properties. Tensile experiments revealed that the elastic modulus increased with a higher PPGDA concentration. Retraction experiments demonstrated increased velocity and acceleration when released from a stretched state with increasing PPGDA concentration. Swelling and deswelling of gels in saline solutions led to a change in mechanical properties and retraction behavior. This study shows that the stretchability and resilience of these hydrogels can be adjusted by changing the concentration of hydrophobic components.

Regulation of Atg8 membrane deconjugation by cysteine proteases in the malaria parasite Plasmodium berghei.

During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance.

Rehabilitation in primary care for an ageing population: a secondary analysis from a scoping review of rehabilitation delivery models.

BACKGROUND: The world population is ageing rapidly. Rehabilitation is one of the most effective health strategies for improving the health and functioning of older persons. An understanding of the current provision of rehabilitation services in primary care (PC) is needed to optimise access to rehabilitation for an ageing population. The objectives of this scoping review are a) to describe how rehabilitation services are currently offered in PC to older persons, and b) to explore age-related differences in the type of rehabilitation services provided. METHODS: We conducted a secondary analysis of a scoping review examining rehabilitation models for older persons, with a focus on PC. Medline and Embase (2015-2022) were searched to identify studies published in English on rehabilitation services for people aged 50 + . Two authors independently screened records and extracted data using the World Health Organization (WHO)'s operational framework, the Primary Health Care Systems (PRIMASYS) approach and the WHO paper on rehabilitation in PC. Data synthesis included quantitative and qualitative analysis. RESULTS: We synthesised data from 96 studies, 88.6% conducted in high-income countries (HICs), with 31,956 participants and identified five models for delivering rehabilitation to older persons in PC: community, home, telerehabilitation, outpatient and eldercare. Nurses, physiotherapists, and occupational therapists were the most common providers, with task-shifting reported in 15.6% of studies. The most common interventions were assessment of functioning, rehabilitation coordination, therapeutic exercise, psychological interventions, and self-management education. Environmental adaptations and assistive technology were rarely reported. CONCLUSIONS: We described how rehabilitation services are currently provided in PC and explored age-related differences in the type of rehabilitation services received. PC can play a key role in assessing functioning and coordinating the rehabilitation process and is also well-placed to deliver rehabilitation interventions. By understanding models of rehabilitation service delivery in PC, stakeholders can work towards developing more comprehensive and accessible services that meet the diverse needs of an ageing population. Our findings, which highlight the role of rehabilitation in healthy ageing, are a valuable resource for informing policy, practice and future research in the context of the United Nations Decade of Healthy Ageing, the Rehab2030 initiative and the recently adopted WHA resolution on strengthening rehabilitation in health systems, but the conclusions can only be applied to HICs and more studies are needed that reflect the reality in low- and middle-income countries.

Incidence of needlestick injury among healthcare workers in western India.

BACKGROUND OBJECTIVES: Injuries occurring from contaminated sharps are a major occupational health hazard. It carries a risk of transmitting blood-borne diseases such as human immunodeficiency virus (HIV), hepatitis B and hepatitis C. Healthcare workers (HCWs), including personnel handling biomedical waste, are at risk. The objective of this study was to determine the incidence and details of needlestick injury (NSI) among HCWs. METHODS: We analyzed data of all HCWs who reported NSI over the past three years. Demographic details, type and source of injury, use of personal protective equipment (PPE), immediate post-exposure measures, hepatitis B vaccination status and HCWs and source's HIV, hepatitis B and hepatitis C serological status were studied. RESULTS: Fifty-six cases of NSI were recorded over three years, accounting for an incidence of 10.4/100 occupied beds per year. Maximum cases (73.2%) occurred between the 20 and 40 yr age group. The distribution among the work category was doctors (37.5%), nursing staff (26.8%), phlebotomy technicians (12.5%), housekeeping/subordinate staff (12.5%) and others (10.7%). Appropriate PPE was donned by 66 per cent of the HCWs. The majority of cases (46.4%) occurred in wards and operating rooms (23.2%). Phlebotomy (35.7%), followed by procedures, such as hemoglucotest (HGT) measurement, intravenous cannula insertion and operative procedures (33.9%), were the most common situation during which HCWs suffered NSI. While 64.2 per cent HCWs were vaccinated for hepatitis B, only 5.4 per cent of the HCWs completed post-exposure anti-retroviral regimen. INTERPRETATION CONCLUSIONS: We conclude that a relative lack of awareness towards preventive measures and inexperience among HCWs may be contributory to high occurrence of NSI events. This study emphasizes upon ensuring active hospital-wide hepatitis B vaccination of all HCWs and supportive therapy to improve compliance towards post-exposure prophylaxis.

Peritoneal dialysis: An effective therapeutic modality in acute kidney injury.

Background: Peritoneal dialysis (PD) as a modality of renal replacement therapy (RRT) in acute kidney injury (AKI), continues to be underused. We present our experience with PD in patients with AKI. Method: The data of patients with AKI requiring RRT were retrospectively analyzed. The primary end point was the adequacy of dialysis, and the secondary end point included hemodynamic stability and procedure-related complications. Result: A total of 32 patients with AKI requiring RRT were included in the study. The mean age and the blood pressure at the time of hospitalization were 65.3 ± 6.73 years and 73.7 ± 8.4 mm Hg, respectively. All the patients required vasopressor support; 26 (81%) patients required ventilator support. RRT was initiated at a mean serum creatinine of 6.24 ± 1.17 mg/dL. Rigid stylet catheter was used in 9 (28.2%) and Tenckhoff catheter in 23 (71.8%) patients. The average daily ultrafiltration and weekly Kt/V achieved were 1701 ± 327 mL and 2.19, respectively; these were significantly higher in survivors. After the initiation of PD, hemodynamic instability was observed in 10 (31.2%) patients. The major therapy-related complication noted was PD peritonitis. Conclusions: In a resource-poor environment, PD is an effective modality of RRT for AKI.

Comparison of unilateral spinal anaesthesia with ultrasound-guided combined sciatic and femoral nerve block in elective arthroscopic knee surgeries.

Background: Arthroscopic knee surgeries are commonly performed orthopaedic procedures, which can be done under unilateral spinal anaesthesia (USA) or ultrasound-guided combined sciatic and femoral nerve block (USFB). However, not many studies have compared both these techniques. Hence this study was undertaken to compare USA and USFB in arthroscopic knee surgeries in terms of time to readiness for discharge (TRD). Methods: Eighty patients were randomised into the USA (n = 40) and USFB groups (n = 40). They were administered either USA or USFB on the affected side. The TRD values were compared. Patients were considered fit for discharge after voiding urine, ambulation and obtaining a visual analogue scale (VAS) score of <3. The maximum time required for any of the three parameters was taken as the TRD for that particular patient. Results: The mean TRD was 595.41 ± 195.69 min in the USA group and 351.86 ± 129.51 min in the USFB group (p < 0.001). The median VAS scores for postoperative pain assessment were lower in the USFB group at 2, 4, 12 and 24 h (p < 0.05). The number of patients requiring rescue analgesia was lower in the USFB group at 6 and 12 h after surgery (p < 0.05). Conclusion: Patients undergoing arthroscopic knee surgeries under USFB have an advantage when it comes to TRD as these patients have comparatively better postoperative analgesia, less requirement of rescue analgesia, early voiding of urine and early ambulation.

Temperature- and strain-dependent transient microstructure and rheological responses of endblock-associated triblock gels of different block lengths in a midblock selective solvent.

Endblock associative ABA gels in midblock selective solvents are attractive due to their easily tunable mechanical properties. Here, we present the effects of A- and B-block lengths on the rheological properties and microstructure of ABA gels by considering three low and one high polymer concentrations. The triblock polymer considered is poly(methyl methacrylate)-poly(n-butyl acrylate)-poly(methyl methacrylate) [PMMA-PnBA-PMMA] and the midblock solvent is 2-ethyl-1-hexanol. The gelation temperature has been found to be strongly dependent on the B-block (PnBA) length, as longer B-blocks facilitate network formation resulting in higher gelation temperature even with lower polymer chain density. Longer A-blocks (PMMA chains) make the endblock association stronger and significantly increase the relaxation time of gels. Temperature-dependent microstructure evolution for the gels with high polymer concentration reveals that the gel microstructure does not change significantly after the gel formation takes place. The dynamic change of microstructure in an applied strain cycle was captured using RheoSAXS experiments. The microstructure orients with the applied strain and the process is reversible in nature, indicating no significant A-block pullout. Our results provide new understandings regarding the temperature and strain-dependent microstructural change of ABA gels in midblock selective solvents.

Correction: Temperature- and strain-dependent transient microstructure and rheological responses of endblock-associated triblock gels of different block lengths in a midblock selective solvent.

Correction for 'Temperature- and strain-dependent transient microstructure and rheological responses of endblock-associated triblock gels of different block lengths in a midblock selective solvent' by Rosa Maria Badani Prado et al., Soft Matter, 2022, 18, 7020-7034, https://doi.org/10.1039/D2SM00567K.

Tracheal intubation using the Airtraq video laryngoscope vs. Macintosh laryngoscope in patients with anticipated difficult intubation.

Background: Various types of laryngoscopes have been invented to ameliorate the laryngoscopic view of the glottis, in normal and difficult airway, which helps anaesthesiologists to secure the airway during anaesthesia. In this prospective study, we aimed to compare the efficacy of the Airtraq video laryngoscope (AVL) and the Macintosh curved-blade laryngoscope (MBL), by using a common clinical assessment tool in patients with modified Mallampati class III and IV. Methods: A total of 60 patients [group A (AVL) and group M (MBL)] with modified Mallampati class III and IV listed for general anaesthesia were included. Each patient was intubated with either of the laryngoscope based on the group allotted. Time taken for tracheal intubation, grade of visualisation of glottis and need for manoeuvres to optimise the glottic view were compared. Results: The degree of the glottic view during successful intubation attempt was easily appreciated in group A (p < 0.0001). Difference in the requirement of manoeuvres for optimising the laryngeal view/assisting in intubation as assessed by manoeuvre score was easily appreciated in group A (p < 0.010). Rise in heart rate and mean arterial pressure 1 and 2 min after intubation was more in group M than in group A (p < 0.0001). No event of any airway trauma, as evidenced by visible trauma to lips or oral mucosa or blood on laryngoscope, was observed with either of the laryngoscope. Conclusion: The novel AVL provides better intubation conditions with greater ease of intubation, better glottic view and lesser haemodynamic alterations during laryngoscopy than MBL.

Dengue infection with warning signs: The 2019 epidemic.

Background: Dengue is a common arthropod-borne viral disease with a variable clinical spectrum, course, and outcome. Method: We conducted a prospective observational study describing the clinical profile of patients hospitalized for dengue with warning signs during the 2019 epidemic. Result: A total of 1349 patients with dengue, including 459 with warning signs, were seen. It predominantly affects the younger subset of both sexes. A higher proportion of females presented with either a platelet count less than 20,000/mm3 or a rapid fall in platelets. Bleeding manifestations of varying severity were observed in one-fifth of them. Severe bleeding was universal at a platelet count less than 10,000/mm3. Platelet transfusion was indicated in 17 (7.3%) males and 25 (11%) females, with females requiring it at a higher platelet count. While hepatic involvement was frequent, others such as myocarditis, encephalitis, and pancreatitis were infrequent. Coinfections observed included malaria, scrub typhus, and enteric fever. The case fatality rate for the patients with dengue and those with warning manifestations was 0.4% and 1.3%, respectively. Conclusions: Dengue epidemics often overwhelm health-care resources. Such epidemics can be successfully managed using a protocolized approach, modified as per the available resources. Platelet transfusions are lifesaving at a count less than 10,000/mm3. With meticulous supportive care, the case fatality rate can be reduced to less than 0.5%.

Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei.

Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host-parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei.

Host biotin is required for liver stage development in malaria parasites.

Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that is the target of several classes of herbicides. Malaria parasites contain a plant-like ACC, and this is the only protein predicted to be biotinylated in the parasite. We found that ACC is expressed in the apicoplast organelle in liver- and blood-stage malaria parasites; however, it is activated through biotinylation only in the liver stages. Consistent with this observation, deletion of the biotin ligase responsible for ACC biotinylation does not impede blood-stage growth, but results in late liver-stage developmental defects. Biotin depletion increases the severity of the developmental defects, demonstrating that parasite and host biotin metabolism are required for normal liver-stage progression. This finding may link the development of liver-stage malaria parasites to the nutritional status of the host, as neither the parasite nor the human host can synthesize biotin.

Pulmonary embolism in coronavirus disease 2019: the silent killer.

BACKGROUND: Pulmonary embolism (PE) has been identified as one of the deadliest complications of coronavirus disease 2019 (COVID-19), especially in patients admitted to the intensive care unit (ICU). Western literature reminds us of the high prevalence of PE in COVID. Here, we report a series of 13 cases of PE diagnosed and managed at our hospital. METHODS: Retrospective analysis of medical records of 13 cases of PE admitted at our hospital from February 1, 2020, to September 31, 2020, were done. Their clinical, laboratory, and radiologic data were assessed in detail. RESULTS: Computed tomography pulmonary arteriography was used to make the diagnosis in eight patients (61.53%), and clinical findings with corroborative ultrasound and laboratory parameters were used to label PE in five patients (38.46%). Five patients were hemodynamically unstable, requiring thrombolysis with recombinant tissue plasminogen activator, and four patients (30.76%) suffered a fatal outcome. CONCLUSION: COVID-19 is a highly prothrombotic state, and all physicians should keep a high vigilance for PE. All hospitalized patients with COVID-19, especially those admitted in ICU, should be on prophylactic anticoagulation and, if there is any worsening, should be started on therapeutic regimen. Patients at the time of discharge should be switched to oral anticoagulation, which should be continued for at least 3-6 months.

A discussion on controversies and ethical dilemmas in prostate cancer screening.

Prostate cancer (PCa) is one of the the most common cancers in men. A blood test called prostate-specific antigen (PSA) has a potential to pick up this cancer very early and is used for screening of this disease. However, screening for prostate cancer is a matter of debate. Level 1 evidence from randomised controlled trials suggests a reduction in cancer-specific mortality from PCa screening. However, there could be an associated impact on quality of life due to a high proportion of overdiagnosis and overtreatment as part of the screening. The US Preventive Services Task Force (USPSTF) in 2012 recommended that PSA-based PCa screening should not to be offered at any age. However, considering the current evidence, USPSTF recently revised its recommendation to offer the PSA test to men aged 55-69 years with shared decision-making, in line with earlier guidelines from the American Cancer Society and the American Urological Association. A shared decision making is necessary since the PSA test could potentially harm an individual. However, the literature suggests that clinicians often neglect a discussion on this issue before ordering the test. This narrative discusses the main controversies regarding PCa screening including the PSA threshold for biopsy, the concept of overdiagnosis and overtreatment, the practical difficulties of active surveillance, the current level 1 evidence on the mortality benefit of screening, and the associated pitfalls. It offers a detailed discussion on the ethics involved in the PSA test and highlights the barriers to shared decision-making and possible solutions.

Proteomic Analysis of Plasmodium Merosomes: The Link between Liver and Blood Stages in Malaria.

The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for antimalarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique data set will allow us to explore key questions about the biology of merosomes and hepatic merozoites.

Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites.

Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical.