Microbial fuel cells using α-amylase-displaying Escherichia coli with starch as fuel.

Escherichia coli JM109 (pGV3-SBA) can assimilate starch by fusing the starch-digesting enzyme α-amylase from Streptococcus bovis NRIC1535 to an OprI' lipoprotein anchor on the cell membrane. This study shows microbial fuel cells (MFCs) development using this recombinant type of E. coli and starch as fuel. We observed the current generation of MFCs with E. coli JM109 (pGV3-SBA) for 120 h. During this period, it consumed 7.1 g/L of starch. A mediator in the form of anthraquinone-2,6-disulfonic acid disodium salt at 0.2, 0.4, and 0.8 mM was added to the MFCs. The highest maximum-current density (271 mA/m2) and maximum-power density (29.3 mW/m2) performances occurred in the 0.4 mM mediator solution. Coulomb yields were calculated as 3.4%, 3.0%, and 3.5% in 1.0, 5.0, and 10.0 g/L of initial starch, respectively. The concentrations of acetic acid, succinic acid, fumaric acid, and ethanol as metabolites were determined. In particular, 38.3 mM of ethanol was produced from 7.1 g/L of starch. This study suggests the use of recombinant E. coli which can assimilate starch present in starch-fueled MFCs. Moreover, it proposes the possibility of gene recombination technology for using wide variety of biomass as fuel and improving MFC's performance.

Production of R- and S-1,2-propanediol in engineered Lactococcus lactis.

1,2-propanediol (1,2-PDO) is a versatile chemical used in multiple manufacturing processes. To date, some engineered and non-engineered microbes, such as Escherichia coli, Lactobacillus buchneri, and Clostridium thermosaccharolyticum, have been used to produce 1,2-PDO. In this study, we demonstrated the production of R- and S-1,2-PDO using engineered Lactococcus lactis. The L- and D-lactic acid-producing L. lactis strains NZ9000 and AH1 were transformed with the plasmid pNZ8048-ppy harboring pct, pduP, and yahK genes for 1,2-PDO biosynthesis, resulting in L. lactis LL1 and LL2, respectively. These engineered L. lactis produced S- and R-1,2-PDO at concentrations of 0.69 and 0.50 g/L with 94.4 and 78.0% ee optical purities, respectively, from 1% glucose after 72 h of cultivation. Both 1% mannitol and 1% gluconate were added instead of glucose to the culture of L. lactis LL1 to supply NADH and NADPH to the 1,2-PDO production pathway, resulting in 75% enhancement of S-1,2-PDO production. Production of S-1,2-PDO from 5% mannitol and 5% gluconate was demonstrated using L. lactis LL1 with a pH-stat approach. This resulted in S-1,2-PDO production at a concentration of 1.88 g/L after 96 h of cultivation. To our knowledge, this is the first report on the production of R- and S-1,2-PDO using engineered lactic acid bacteria.

Digital triage: Novel strategies for population health management in response to the COVID-19 pandemic.

The COVID-19 pandemic has created unique challenges for the U.S. healthcare system due to the staggering mismatch between healthcare system capacity and patient demand. The healthcare industry has been a relatively slow adopter of digital innovation due to the conventional belief that humans need to be at the center of healthcare delivery tasks. However, in the setting of the COVID-19 pandemic, artificial intelligence (AI) may be used to carry out specific tasks such as pre-hospital triage and enable clinicians to deliver care at scale. Recognizing that the majority of COVID-19 cases are mild and do not require hospitalization, Partners HealthCare (now Mass General Brigham) implemented a digitally-automated pre-hospital triage solution to direct patients to the appropriate care setting before they showed up at the emergency department and clinics, which would otherwise consume resources, expose other patients and staff to potential viral transmission, and further exacerbate supply-and-demand mismatching. Although the use of AI has been well-established in other industries to optimize supply and demand matching, the introduction of AI to perform tasks remotely that were traditionally performed in-person by clinical staff represents a significant milestone in healthcare operations strategy.

Engineering Escherichia coli for Direct Production of 1,2-Propanediol and 1,3-Propanediol from Starch.

Diols are versatile chemicals used for multiple manufacturing products. In some previous studies, Escherichia coli has been engineered to produce 1,2-propanediol (1,2-PDO) and 1,3-propanediol (1,3-PDO) from glucose. However, there are no reports on the direct production of these diols from starch instead of glucose as a substrate. In this study, we directly produced 1,2-PDO and 1,3-PDO from starch using E. coli engineered for expressing a heterologous α-amylase, along with the expression of 1,2-PDO and 1,3-PDO synthetic genes. For this, the recombinant plasmids, pVUB3-SBA harboring amyA gene for α-amylase production, pSR5 harboring pct, pduP, and yahK genes for 1,2-PDO production, and pSR8 harboring gpd1-gpp2, dhaB123, gdrAB, and dhaT genes for 1,3-PDO production, were constructed. Subsequently, E. coli BW25113 (ΔpflA) and BW25113 strains were transformed with pVUB3-SBA, pSR5, and/or pSR8. Using these transformants, direct production of 1,2-PDO and 1,3-PDO from starch was demonstrated under microaerobic condition. As a result, the maximum production titers of 1,2-PDO and 1,3-PDO from 1% glucose as a sole carbon source were 13 mg/L and 150 mg/L, respectively. The maximum production titers from 1% starch were similar levels (30 mg/L 1,2-PDO and 120 mg/L 1,3-PDO). These data indicate that starch can be an alternative carbon source for the production of 1,2-PDO and 1,3-PDO in engineered E. coli. This technology could simplify the upstream process of diol bioproduction.

Usage Patterns of a Web-Based Palliative Care Content Platform (PalliCOVID) During the COVID-19 Pandemic.

CONTEXT: The COVID-19 pandemic has highlighted the essential role of palliative care to support the delivery of compassionate, goal-concordant patient care. We created the Web-based application, PalliCOVID (https://pallicovid.app/), in April 2020 to provide all clinicians with convenient access to palliative care resources and support. PalliCOVID features evidence-based clinical guidelines, educational content, and institutional protocols related to palliative care for COVID-19 patients. It is a publicly available resource accessible from any mobile device or desktop computer that provides clinicians with access to palliative care guidance across a variety of care settings, including the emergency department, hospital ward, intensive care unit, and primary care practice. OBJECTIVE: The primary objective of this study was to evaluate usage patterns of PalliCOVID to understand user behavior in relation to this palliative care content platform during the period of the local peak of COVID-19 infection in Massachusetts. METHODS: We retrospectively analyzed deidentified usage data collected by Google Analytics from the first day of PalliCOVID's launch on April 7, 2020, until May 1, 2020, the time period that encompassed the local peak of the COVID-19 surge in Massachusetts. User access data were collected and summarized by using Google Analytics software that had been integrated into the PalliCOVID Web application. RESULTS: A total of 2042 users accessed PalliCOVID and viewed 4637 pages from April 7 to May 1, 2020. Users spent an average of 2 minutes and 6 seconds per session. Eighty-one percent of users were first-time visitors, while the remaining 19% were return visitors. Most users accessed PalliCOVID from the United States (87%), with a large proportion of users coming from Boston and the surrounding cities (32% of overall users). CONCLUSIONS: PalliCOVID is one example of a scalable digital health solution that can bring palliative care resources to frontline clinicians. Analysis of PalliCOVID usage patterns has the potential to inform the improvement of the platform to better meet the needs of its user base and guide future dissemination strategies. The quantitative data presented here, although informative about user behavior, should be supplemented with future qualitative research to further define the impact of this tool and extend our ability to deliver clinical care that is compassionate, rational, and well-aligned with patients' values and goals.

Development of a Palliative Care Toolkit for the COVID-19 Pandemic.

The Coronavirus disease 2019 (COVID-19) pandemic has led to high numbers of critically ill and dying patients in need of expert management of dyspnea, delirium, and serious illness communication. The rapid spread of severe acute respiratory syndrome-Coronavirus-2 creates surges of infected patients requiring hospitalization and puts palliative care programs at risk of being overwhelmed by patients, families, and clinicians seeking help. In response to this unprecedented need for palliative care, our program sought to create a collection of palliative care resources for nonpalliative care clinicians. A workgroup of interdisciplinary palliative care clinicians developed the Palliative Care Toolkit, consisting of a detailed chapter in a COVID-19 online resource, a mobile and desktop Web application, one-page guides, pocket cards, and communication skills training videos. The suite of resources provides expert and evidence-based guidance on symptom management including dyspnea, pain, and delirium, as well as on serious illness communication, including conversations about goals of care, code status, and end of life. We also created a nurse resource hotline staffed by palliative care nurse practitioners and virtual office hours staffed by a palliative care attending physician. Since its development, the Toolkit has helped us disseminate best practices to nonpalliative care clinicians delivering primary palliative care, allowing our team to focus on the highest-need consults and increasing acceptance of palliative care across hospital settings.

Stable mutants of restriction-deficient/modification-proficient Bacillus subtilis 168: hub strains for giant DNA engineering.

Bacillus subtilis 168 has been explored as a platform for the synthesis and transmission of large DNA. Two inherent DNA incorporation systems, natural transformation and pLS20-based conjugation transfer, enable rapid handling of target DNA. Both systems are affected by the Bsu restriction-modification system that recognizes and cleaves unmethylated XhoI sites, limiting the choice of target DNA. We constructed B. subtilis 168 with stable mutation for restriction-deficient and modification-proficient (r-m+). It was demonstrated that the r-m+ strains can incorporate and transfer synthesized DNA with multiple XhoI sites. These should be of value as hub strains to integrate and disseminate giant DNA between B. subtilis 168 derivatives.

Efficient delivery of large DNA from Escherichia coli to Synechococcus elongatus PCC7942 by broad-host-range conjugal plasmid pUB307.

Synechococcus elongatus PCC7942, a cyanobacterium that uses light and carbon dioxide to grow, has a high ability to incorporate DNA by transformation. To assess the effective delivery of large DNA in plasmid form, we cloned the endogenous plasmid pANL (46.4 kbp) into a BAC vector of Escherichia coli. The plasmid p38ANL (54.3 kbp) replaced the native plasmid. To assess the delivery of larger DNA into PCC7942, p38ANL was fused to the broad-host-range conjugal transfer plasmid pUB307IP (53.5 kbp). The resulting plasmid pUB307IP501 (107.9 kbp) was transmitted from E. coli to PCC7942 by simple mixing of donor and recipient cultures. PCC7942 transcipients possessed only pUB307IP501, replacing the preexisting pANL. In contrast, the pUB307IP501 plasmid was unable to transform PCC7942, indicating that natural transformation of DNA may be restricted by size limitations. The ability to deliver large DNA by conjugation may lead to genetic engineering in PCC7942.

Detection and genotyping of Cryptosporidium spp. in large Japanese field mice, Apodemus speciosus.

The genus Cryptosporidium, which is an obligate intracellular parasite, infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Wild rodents are naturally infected with zoonotic Cryptosporidium; thus, they are potential reservoirs of the parasites. Mice are common rodents frequently found in agricultural areas and have many opportunities to contact other wild animals, livestock, and humans. Irrespective of the potential risk, there are few epidemiologic studies of Cryptosporidium in wild mice because of their low economic importance and the difficulty in conducting surveys. Hence, the species and genotypes of Cryptosporidium in wild mice living around various areas remain unclear. We investigated the species and genotype distribution and prevalence of Cryptosporidium in the large Japanese field mouse (Apodemus speciosus) in an agricultural site in Osaki, Miyagi Prefecture, Japan. In total, 15 mice were captured and examined in this study. By microscopic analysis, only one mouse (JFM 3) was determined to be Cryptosporidium-positive, while the parasite were detected in four mice (JFM 3, 6, 10, and 15) by a molecular approach using partial SSU rRNA gene sequences. Based on nucleotide sequence and phylogenetic analysis, the Cryptosporidium isolates were identified as C. ubiquitum (from JFM 10) and C. muris (from JFM 3 and 6). In contrast, the Cryptosporidium in JFM 15 was not identified as a known species or genotype and is therefore proposed as a novel genotype; the Naruko genotype. More molecular data are necessary to elucidate the taxonomic identity of this novel Cryptosporidium genotype. The C. muris Japanese field mouse genotypes showed marked divergence compared to that in a previous report. The large Japanese field mouse might thus represent a reservoir of multiple Cryptosporidium spp.

Molecular characterization of Cryptosporidium in pigs in central Vietnam.

Little information is available on the epidemiology of Cryptosporidium in pigs in central Vietnam. The aims of this study were to investigate the prevalence and to characterize the genotype distribution of Cryptosporidium isolates in pigs in this region. A total of 193 pig fecal samples were screened for the presence of Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method, and 28 (overall prevalence 14.5 %) were identified as positive by microscopic observation. Positive samples were further analyzed by polymerase chain reaction amplification and sequencing. Genetic identification based on the 18S ribosomal RNA and 70 kDa heat shock protein genes revealed that pigs in Vietnam are infected with two species/genotypes (Cryptosporidium suis and Cryptosporidium pig genotype II). This study is the first molecular characterization of Cryptosporidium in pigs in Vietnam. The presence of these host-adapted species/genotypes suggests that pigs may not pose a significant public health risk in this area. More extensive studies are necessary to ascertain the zoonotic potential of Cryptosporidium in porcine hosts in Vietnam.

Molecular characterization of Cryptosporidium in native beef calves in central Vietnam.

The aims of this study were to investigate the prevalence of Cryptosporidium and to characterize the genotype distribution of Cryptosporidium isolates in native beef calves 2-6 months old in Dac Lac province, central Vietnam. The presence of Cryptosporidium oocysts was determined using the modified Ziehl-Neelsen staining method. The overall prevalence on the sample and herd levels were 18.9% (44/232) and 50% (20/40), respectively. Genotyping based on PCR and sequence analysis of the 18 S rRNA gene revealed occurrence of the two nonzoonotic species Cryptosporidium ryanae and Cryptosporidium bovis, with the former as a dominant species in the animals. The absence of the zoonotic species Cryptosporidium parvum in calves examined suggests that the native beef calves 2-6 months old in the study area are unlikely to contribute to human cryptosporidiosis transmission.

Molecular characterization of Cryptosporidium spp. in grazing beef cattle in Japan.

Cattle are major hosts of Cryptosporidium spp. Cryptosporidiosis in neonatal calves is associated with retarded growth, weight loss and calf mortality, and zoonotic infections in humans. In many areas, cow-calf glazing system is an important beef cattle rearing method with distinct advantages in terms of cost and the labor required. However, few epidemiologic studies of Cryptosporidium spp. have been conducted in this system, especially using molecular diagnostic tools. To understand the transmission of Cryptosporidium spp. in a grazing system, we followed cryptosporidiosis on a grazing farm in Osaki City, Miyagi Prefecture, in northwest Japan for one year. Fecal samples were collected from Japanese Black and Japanese Shorthorn cattle and examined by PCR-RFLP and sequence analyses. Of 113 fecal samples collected in October 2010, 23 (20%) were positive for Cryptosporidium, including 15 samples (13%) having C. bovis, 6 (5%) having C. ryanae, and 2 (2%) having mixed infections of both species. Additionally, C. bovis or C. ryanae was detected on all other sampling dates involving smaller numbers of animals. The infection rate of C. bovis was significantly different among age groups, and calve-to-calve infection might be the major route of cryptosporidiosis transmission in beef cattle. Interestingly, one animal had C. bovis infection or re-infection for one year. Our results suggest that C. bovis and C. ryanae are distributed in Japan, but might have low level of detection in grazing beef cattle.