Leveraging mathematical models for optimizing filter utility at manufacturing scale.

In the production of biopharmaceuticals depth filters followed by sterile filters are often employed to remove residual cell debris present in the feed stream. In the back drop of a global pandemic, supply chains associated with the production of biopharmaceuticals have been constrained. These constraints have limited the available amount of depth filters for the manufacture of biologics. This has placed manufacturing facilities in a difficult position having to choose between running processes with reduced number of depth filters and risking a failed batch or the prospect of plants going into temporary shutdown until the depth filter resources are replenished. This communication describes a modeling based method that leverages manufacturing scale filtration data to predict the depth filter performance with a reduced number of filters and an increased operational flux. This method can be used to quantify the acceptable level of area reduction before which the filtration process performance is affected. This enables facilities to manage their filter inventory avoiding potential plant shutdowns and reduces the risks of negative depth filter performance.

A scale-down mimic for mapping the process performance of centrifugation, depth and sterile filtration.

In the production of biopharmaceuticals disk-stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot-scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale-down approach based upon the use of a shear device and a bench-top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large-scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934-1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Prevalence of Empiric Antibacterial Therapy, Community-Acquired Bacterial Superinfection, and Antibiotic-Associated Adverse Reactions among Patients with COVID-19 Pneumonia Admitted in Makati Medical Center from March 2020 to March 2021.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus which was first discovered in Wuhan, China. Being a viral illness, antibacterial agents theoretically have no role in patients with pure COVID-19 infection. However, like any viral illness, concomitant bacterial infection may occur. The dilemma of starting an antibacterial agent in a COVID-19 patient remains a debate since the use of antibacterial agents may pose a risk of developing antibiotic-associated adverse events such as the emergence of drug-resistant pathogens and other antibiotic-associated drug toxicity. The primary objective of the study is to determine the amount of empiric antibacterial prescription done by physicians among admitted patients with COVID-19 infection as well as the trend of antibiotic prescription by physicians during the past 12 months of the COVID-19 pandemic. The secondary objective was to determine and quantify antibiotic-associated adverse effects. MATERIALS AND METHODS: This is a retrospective cohort study wherein charts of patients admitted for COVID-19 last March 2020 to March 2021 were reviewed and analyzed. Empiric antibiotic prescription during the first 48 hours of admission was noted as well as the proportion of concomitant bacterial infections. Development of antibiotic-associated adverse events such as the development of the multidrug-resistant organism and fungal infections, Clostridiodes difficile and/or gastrointestinal side effects, and hypersensitivity reactions were also noted. RESULTS: Results showed that among the 895 patients with COVID-19 admitted, 533 (59.6%) patients were started antibiotics during the first 48 hours of admission. Among those patients who are started with antibacterial therapy during the first 48 hours of admission, 60 (15.3%) patients had bacterial coinfection. The prevalence of antibiotic-associated adverse events was 46.9%, the most common of which was gastrointestinal reactions. The overall mortality rate of the patients given antibiotics was 18.8%. The median length of hospital stay was 11 days. CONCLUSION: Community-acquired bacterial infections in COVID-19 patients admitted during the study period were low while empiric antibiotic prescription was high especially during the first few months of the pandemic, especially during the surge. Antibiotic-related adverse effects were high. There was a noted decreasing trend of antibiotic prescription during the latter part of the pandemic when new developments in COVID-19 treatment were discovered. All in all, routine antibacterial prescription in patients with COVID-19 is not recommended given the real-world data in this study.

High TB burden and low notification rates in the Philippines: The 2016 national TB prevalence survey.

SETTING: The 3rd national tuberculosis (TB) survey in the Philippines in 2007 reported a significant decline in the prevalence of TB. Since then, more significant investments for TB control have been made, yet TB burden estimates from routine surveillance data remain relatively stable. OBJECTIVE: To estimate the prevalence of bacteriologically confirmed pulmonary TB in the Philippines amongst individuals aged ≥15 years in 2016. DESIGN: In March-December 2016, we conducted a population-based survey with stratified, multi-stage cluster sampling of residents in 106 clusters aged ≥15 years. Survey participants were screened for TB by symptom-based interview and digital chest X-ray. Those with cough ≥2 weeks and/or haemoptysis and/or chest X-ray suggestive of TB were requested to submit 2 sputum specimens for Xpert MTB/RIF, direct sputum smear microscopy using LED fluorescent microscopy, and mycobacterial solid culture (Ogawa method). Bacteriologically confirmed pulmonary TB was defined as MTB culture positive and/or Xpert positive. RESULTS: There were 46,689 individuals interviewed, and 41,444 (88.8%) consented to a chest X-ray. There were 18,597 (39.8%) eligible for sputum examination and 16,242 (87.3%) submitted at least one specimen. Out of 16,058 sputum-eligible participants, 183 (1.1%) were smear-positive. There were 466 bacteriologically confirmed TB cases: 238 (51.1%) Xpert positive, 69 (14.8%) culture positive, and 159 (34.1%) positive by both Xpert and culture. The estimated TB prevalence per 100,000 population aged ≥15 years was 434 (95% CI: 350-518) for smear-positive TB, and 1,159 (95% CI: 1,016-1,301) for bacteriologically confirmed TB. CONCLUSION: This nationally representative survey found that the TB burden in the Philippines in 2016 was higher than estimated from routine TB surveillance data. There was no evidence of a decline in smear and culture positive TB from the 2007 survey despite significant investments in TB control. New strategies for case-finding and patient-centered care must be intensified and expanded.

Tuning cell behavior with nanoparticle shape.

We investigated how the shape of polymeric vesicles, made by the exact same material, impacts the replication activity and metabolic state of both cancer and non-cancer cell types. First, we isolated discrete geometrical structures (spheres and tubes) from a heterogeneous sample using density-gradient centrifugation. Then, we characterized the cellular internalization and the kinetics of uptake of both types of polymersomes in different cell types (either cancer or non-cancer cells). We also investigated the cellular metabolic response as a function of the shape of the structures internalized and discovered that tubular vesicles induce a significant decrease in the replication activity of cancer cells compared to spherical vesicles. We related this effect to the significant up-regulation of the tumor suppressor genes p21 and p53 with a concomitant activation of caspase 3/7. Finally, we demonstrated that combining the intrinsic shape-dependent effects of tubes with the delivery of doxorubicin significantly increases the cytotoxicity of the system. Our results illustrate how the geometrical conformation of nanoparticles could impact cell behavior and how this could be tuned to create novel drug delivery systems tailored to specific biomedical application.

Refractory Burkholderia cepacia bacteraemia from a consolidation pneumonia lasting more than 7 weeks, successfully treated with systemic antibiotics and nebulised meropenem.

We present a case of a 55-year-old Filipino man who was transferred from another institution where he was recently diagnosed with Crohn's disease but not started on any immunosuppressants. He underwent laparoscopic cholecystectomy with T-tube placement a few weeks prior to admission. On workup, abdominal CT scan was unremarkable, but blood cultures on the third hospital day grew Burkholderia cepacia Antibiotic regimen was shifted to ceftazidime and levofloxacin. The bacteraemia and febrile episodes persisted despite removal of the central line and T tube. White blood cell scan and chest CT scan showed left-sided consolidation pneumonia. Blood cultures continued to grow B. cepacia despite shifting to meropenem and trimethoprim-sulfamethoxazole. Meropenem nebulisation at 250 mg every 12 hours was added to the regimen on the third week then oral minocycline was added on the fourth week due to persistence of bacteraemia. He subsequently developed a small vegetation on the aortic valve, so amikacin was added. Fever lysed on the sixth week, but the B. cepacia bacteraemia persisted, clearing only on the 51st hospital day. The patient was discharged with a plan to continue antibiotics, including meropenem nebulisation, for 6 more weeks. On follow-up, the patient had no recurrence of fever. There was also resolution of consolidation on chest CT scan and disappearance of vegetation on echocardiography.

Manufacturing of Proteins and Antibodies: Chapter Downstream Processing Technologies.

Cell harvesting is the separation or retention of cells and cellular debris from the supernatant containing the target molecule Selection of harvest method strongly depends on the type of cells, mode of bioreactor operation, process scale, and characteristics of the product and cell culture fluid. Most traditional harvesting methods use some form of filtration, centrifugation, or a combination of both for cell separation and/or retention. Filtration methods include normal flow depth filtration and tangential flow microfiltration. The ability to scale down predictably the selected harvest method helps to ensure successful production and is critical for conducting small-scale characterization studies for confirming parameter targets and ranges. In this chapter we describe centrifugation and depth filtration harvesting methods, share strategies for harvest optimization, present recent developments in centrifugation scale-down models, and review alternative harvesting technologies.

Incorporation of the HEART Score Into a Low-risk Chest Pain Pathway to Safely Decrease Admissions.

Chest pain can be a challenging complaint to manage in the emergency department. A missed diagnosis can result in significant morbidity or mortality, whereas avoidable testing and hospitalizations can lead to increased health care costs, contribute to hospital crowding, and increase risks to patients. The HEART score is a validated decision aid to identify patients at low risk for acute coronary syndrome who can be safely discharged without admission or objective cardiac testing. In the largest and one of the longest studies to date (N = 31,060; 30 months), we included the HEART score into a larger, newly developed low-risk chest pain decision pathway, using a retrospective observational pre/post study design with the objective of safely lowering admissions. The modified HEART score calculation tool was incorporated in our electronic medical record. A significant increase in discharges of low-risk chest pain patients (relative increase of 21%; p < 0.0001) in the postimplementation period was observed with no significant difference in the rates of major adverse cardiac events between the pre and post periods. There was a decrease in the amount of return admissions for 30 days (4.65% fewer; p = 0.009) and 60 days (3.78% fewer; p = 0.020). No significant difference in length of stay was observed for patients who were ultimately discharged. A 64% decrease in monthly coronary computed tomography angiograms was observed in the post period (p < 0.0001). These findings support the growing consensus in the literature that the adoption of the HEART pathway or similar protocols in emergency departments, including at large and high-volume medical institutions, can substantially benefit patient care and reduce associated health care costs.

The role of the two splice variants and extranuclear pathway on Ki-67 regulation in non-cancer and cancer cells.

Ki-67 is a nuclear protein that has been used in cancer diagnostic because of its specific cell-cycle dependent expression profile. After quantifying and characterising the expression level of Ki-67, as a function of the cell cycle, we found out that the two main splice variants of the protein (i.e. α and ß) are differently regulated in non-cancerous and cancerous cells both at mRNA and protein level. We were able to correlate the presence of the α variant of the protein with the progression through the interphase of cell cycle. We also observed that the different expression profiles correspond to different degradation pathways for non-cancerous and cancerous cells. Furthermore, Ki-67 is continuously regulated and degraded via proteasome system in both cell types, suggesting an active control of the protein. However we also observed a putative extranuclear elimination pathway of Ki-67 where it is transported to the Golgi apparatus. Our evidence in the different expression of the splice variants may represent a milestone for the development of new targets for cancer diagnostic and prognostic. Additionally, the unexpected extranuclear elimination of Ki-67 strongly suggests that this protein must be looked at also outside of the "nuclear box", as thought to date.

An automated laboratory-scale methodology for the generation of sheared mammalian cell culture samples.

Continuous disk-stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing-scale. The use of scale-down methods to characterise disk-stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot-scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines. This paper describes an alternative and easy-to-assemble automated capillary-based methodology to generate levels of EDRs consistent with those found in a continuous disk-stack centrifuge. Variations in EDR were achieved through changes in capillary internal diameter and the flow rate of operation through the capillary. The EDRs found to match the levels of shear in the feed zone of a pilot-scale centrifuge using the experimental method developed in this paper (2.4×105 W/Kg) are consistent with those obtained through previously published computational fluid dynamic (CFD) studies (2.0×105 W/Kg). Furthermore, this methodology can be incorporated into existing scale-down methods to model the process performance of continuous disk-stack centrifuges. This was demonstrated through the characterisation of culture hold time, culture temperature and EDRs on centrate quality.

Chemotactic synthetic vesicles: Design and applications in blood-brain barrier crossing.

In recent years, scientists have created artificial microscopic and nanoscopic self-propelling particles, often referred to as nano- or microswimmers, capable of mimicking biological locomotion and taxis. This active diffusion enables the engineering of complex operations that so far have not been possible at the micro- and nanoscale. One of the most promising tasks is the ability to engineer nanocarriers that can autonomously navigate within tissues and organs, accessing nearly every site of the human body guided by endogenous chemical gradients. We report a fully synthetic, organic, nanoscopic system that exhibits attractive chemotaxis driven by enzymatic conversion of glucose. We achieve this by encapsulating glucose oxidase alone or in combination with catalase into nanoscopic and biocompatible asymmetric polymer vesicles (known as polymersomes). We show that these vesicles self-propel in response to an external gradient of glucose by inducing a slip velocity on their surface, which makes them move in an extremely sensitive way toward higher-concentration regions. We finally demonstrate that the chemotactic behavior of these nanoswimmers, in combination with LRP-1 (low-density lipoprotein receptor-related protein 1) targeting, enables a fourfold increase in penetration to the brain compared to nonchemotactic systems.

Prevalence of Empiric Antibacterial Therapy, Community-Acquired Bacterial Superinfection, and Antibiotic-Associated Adverse Reactions among Patients with COVID-19 Pneumonia Admitted in Makati Medical Center from March 2020 to March 2021 / 감염과화학요법

Background@#Coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus which was first discovered in Wuhan, China. Being a viral illness, antibacterial agents theoretically have no role in patients with pure COVID-19 infection. However, like any viral illness, concomitant bacterial infection may occur. The dilemma of starting an antibacterial agent in a COVID-19 patient remains a debate since the use of antibacterial agents may pose a risk of developing antibiotic-associated adverse events such as the emergence of drug-resistant pathogens and other antibiotic-associated drug toxicity. The primary objective of the study is to determine the amount of empiric antibacterial prescription done by physicians among admitted patients with COVID-19 infection as well as the trend of antibiotic prescription by physicians during the past 12 months of the COVID-19 pandemic. The secondary objective was to determine and quantify antibiotic-associated adverse effects. @*Materials and Methods@#This is a retrospective cohort study wherein charts of patients admitted for COVID-19 last March 2020 to March 2021 were reviewed and analyzed. Empiric antibiotic prescription during the first 48 hours of admission was noted as well as the proportion of concomitant bacterial infections. Development of antibiotic-associated adverse events such as the development of the multidrug-resistant organism and fungal infections, Clostridiodes difficile and/or gastrointestinal side effects, and hypersensitivity reactions were also noted. @*Results@#Results showed that among the 895 patients with COVID-19 admitted, 533 (59.6%) patients were started antibiotics during the first 48 hours of admission. Among those patients who are started with antibacterial therapy during the first 48 hours of admission, 60 (15.3%) patients had bacterial coinfection. The prevalence of antibiotic-associated adverse events was 46.9%, the most common of which was gastrointestinal reactions. The overall mortality rate of the patients given antibiotics was 18.8%. The median length of hospital stay was 11 days. @*Conclusion@#Community-acquired bacterial infections in COVID-19 patients admitted during the study period were low while empiric antibiotic prescription was high especially during the first few months of the pandemic, especially during the surge. Antibiotic-related adverse effects were high. There was a noted decreasing trend of antibiotic prescription during the latter part of the pandemic when new developments in COVID-19 treatment were discovered. All in all, routine antibacterial prescription in patients with COVID-19 is not recommended given the real-world data in this study.

Variation in outpatient emergency department utilization in Texas Medicaid: a state-level framework for finding "superutilizers".

BACKGROUND: Very frequent outpatient emergency department (ED) use-so called "superutilization"-at the state level is not well-studied. To address this gap, we examined frequent ED utilization in the largest state Medicaid population to date. METHODS: Using Texas Medicaid (the third largest in the USA) claims data, we examined the variability in expenditures, sociodemographics, comorbidities, and persistence across seven levels of ED utilization/year (i.e., 1, 2, 3-4, 5-6, 7-9, 10-14, and ≥ 15 visits). We classified visits into emergent and non-emergent categories using the most recent New York University algorithm. RESULTS: Thirty-one percent (n = 346,651) of Texas Medicaid adult enrollees visited the ED at least once in 2014. Enrollees with ≥ 3 ED visits accounted for 8.5% of all adult patients, 60.4% of the total ED visits, and 62.1% of the total ED expenditures. Extremely frequent ED users (≥ 10 ED visits) represented < 1% of all users but accounted for 15.5% of all ED visits and 17.4% of the total ED costs. The proportions of ED visits classified as non-emergent or emergent, but primary care treatable varied little as ED visits increased. Overall, approximately 13% of ED visits were considered not preventable or avoidable. CONCLUSIONS: The Texas Medicaid population has a substantial burden of chronic disease with only modest increases in substance use and mental health diagnoses as annual visits increase. Understanding the characteristics that lead to frequent ED use is vital to developing strategies and Medicaid policy to reduce high utilization.

Molecular engineering of polymersome surface topology.

Biological systems exploit self-assembly to create complex structures whose arrangements are finely controlled from the molecular to mesoscopic level. We report an example of using fully synthetic systems that mimic two levels of self-assembly. We show the formation of vesicles using amphiphilic copolymers whose chemical nature is chosen to control both membrane formation and membrane-confined interactions. We report polymersomes with patterns that emerge by engineering interfacial tension within the polymersome surface. This allows the formation of domains whose topology is tailored by chemical synthesis, paving the avenue to complex supramolecular designs functionally similar to those found in viruses and trafficking vesicles.

Polyethylene glycol (PEG)-dendron phospholipids as innovative constructs for the preparation of super stealth liposomes for anticancer therapy.

Pegylation of nanoparticles has been widely implemented in the field of drug delivery to prevent macrophage clearance and increase drug accumulation at a target site. However, the shielding effect of polyethylene glycol (PEG) is usually incomplete and transient, due to loss of nanoparticle integrity upon systemic injection. Here, we have synthesized unique PEG-dendron-phospholipid constructs that form super stealth liposomes (SSLs). A ß-glutamic acid dendron anchor was used to attach a PEG chain to several distearoyl phosphoethanolamine lipids, thereby differing from conventional stealth liposomes where a PEG chain is attached to a single phospholipid. This composition was shown to increase liposomal stability, prolong the circulation half-life, improve the biodistribution profile and enhance the anticancer potency of a drug payload (doxorubicin hydrochloride).

Clonal relatedness is a predictor of spontaneous multidrug efflux pump gene overexpression in Staphylococcus aureus.

Increased expression of genes encoding multidrug resistance efflux pumps (MDR-EPs) contributes to antimicrobial agent and biocide resistance in Staphylococcus aureus. Previously identified associations between norA overexpression and spa type t002 meticillin-resistant S. aureus (MRSA), and a similar yet weaker association between mepA overexpression and type t008 meticillin-susceptible S. aureus (MSSA), in clinical isolates are suggestive of clonal dissemination. It is also possible that related strains are prone to mutations resulting in overexpression of specific MDR-EP genes. Exposure of non-MDR-EP-overexpressing clinical isolates to biocides and dyes can select for MDR-EP-overexpressing mutants. spa types t002 and t008 isolates are predominated by multilocus sequencing typing sequence types (STs) 5 and 8, respectively. In this study, non-MDR-EP gene-overexpressing clinical isolates (MRSA and MSSA) representing ST5 and ST8 were subjected to single exposures of ethidium bromide (EtBr) to select for EtBr-resistant mutants. Measurements of active EtBr transport among mutants were used to demonstrate an efflux-proficient phenotype. Using quantitative reverse-transcription PCR, it was found that EtBr-resistant mutants of ST5 and ST8 parental strains predominantly overexpressed mepA (100%) and mdeA (83%), respectively, regardless of meticillin sensitivity. Associations between clonal lineage and MDR-EP gene overexpression differed from those previously observed and suggest the latter is due to clonal spread of efflux-proficient strains. The predilection of in vitro-selected mutants of related strains to overexpress the same MDR-EP gene indicates the presence of a consistent mutational process.

Live cell imaging of membrane/cytoskeleton interactions and membrane topology.

We elucidate the interaction between actin and specific membrane components, using real time live cell imaging, by delivering probes that enable access to components, that cannot be accessed genetically. We initially investigated the close interplay between Phosphatidylinositol 4,5-bisphosphate (PIP2) and the F-actin network. We show that, during the early stage of cell adhesion, PIP2 forms domains within the filopodia membrane. We studied these domains alongside cell spreading and observed that these very closely follow the actin tread-milling. We show that this mechanism is associated with an active transport of PIP2 rich organelles from the cell perinuclear area to the edge, along actin fibers. Finally, mapping other phospholipids and membrane components we observed that the PIP2 domains formation is correlated with sphingosine and cholesterol rafts.

Prophylaxis against hepatitis B reactivation among patients with lymphoma receiving rituximab.

Hepatitis B virus (HBV) reactivation occurs commonly in patients with non-Hodgkin lymphoma, receiving rituximab-based therapeutic regimen. Guidelines from different organizations are not all in agreement with regard to screening and antiviral prophylaxis, given the limited evidence. Antiviral prophylaxis has been recommended for all HBV surface antigen-positive patients. Evidence for benefit from prophylaxis has recently been recognized among HBV surface antigen-negative/HBV core antibody-positive patients. Incidence of HBV reactivation varies in different geographical locations and the decision to start antiviral prophylaxis should consider local incidence data. Given the increased rates of rituximab-associated HBV reactivation and potentially fatal outcome, it is prudent that all patients who receive rituximab should be screened for HBV markers.

In vivo contextual requirements for UAG translation as pyrrolysine.

Pyrrolysine and selenocysteine have infiltrated natural genetic codes via the translation of canonical stop codons. UGA translation as selenocysteine is absolutely dependent on message context. Here we describe the first experimental examination of contextual requirements for UAG translation as pyrrolysine. A hexahistidine-tagged Methanosarcina barkeri mtmB1 gene, encoding monomethylamine methyltransferase MtmB1, was introduced into Methanosarcina acetivorans. Host mtmB expression was minimized by growth on methanol and recombinant mtmB1 products monitored by anti-MtmB and anti-hexahistidine immunoblotting. UAG translation was not compromised, as recombinant MtmB1 was 1% of cellular protein with only trace UAG-terminated mtmB1 product detectable. Untranslated regions flanking mtmB1 were not required for UAG translation, but loss of a downstream pyrrolysine insertion sequence (PYLIS) significantly increased the UAG-termination product of mtmB1 and decreased the UAG-translation product, which nonetheless contained pyrrolysine. An in-frame UAG within a bacterial uidA transcript was translated in the methanogen as pyrrolysine with 20% efficiency, suggesting UAG translation in the absence of evolved context. However, predominant UAG-directed termination with enhancement of UAG translation by the PYLIS appears analogous to cis-acting elements for UGA translation as selenocysteine, although different mechanisms may underlie these recoding events.