Design and engineering of artificial biosynthetic pathways—where do we stand and where do we go?

Design and engineering of artificial biosynthetic pathways—where do we stand and where do we go?

Microbial production of commodity and specialty chemicals has the potential to decrease our reliance on fossil fuels and reduce the accompanying environmental effects. The scope of traditional biomanufacturing can be greatly enhanced by constructing new-to-nature pathways toward target chemicals. Here, we outline the principles of artificial pathway design and engineering, highlight notable examples and provide an outlook on its future.


The production of commodity and specialty chemicals relies heavily on fossil fuels. The negative impact of this dependency on our environment and climate has spurred a rising demand for more sustainable methods to obtain such chemicals from renewable resources. Herein, biotransformations of these renewable resources facilitated by enzymes or (micro)organisms have gained significant attention, since they can occur under mild conditions and reduce waste. These biotransformations typically leverage natural metabolic processes, which limits the scope and production capacity of such processes. In this mini-review, we provide an overview of advancements made in the past 5 years to expand the repertoire of biotransformations in engineered microorganisms. This ranges from redesign of existing pathways driven by retrobiosynthesis and computational design to directed evolution of enzymes and de novo pathway design to unlock novel routes for the synthesis of desired chemicals. We highlight notable examples of pathway designs for the production of commodity and specialty chemicals, showcasing the potential of these approaches. Lastly, we provide an outlook on future pathway design approaches.

Dried blood spot based biomarkers in the Health and Retirement Study: 2006 to 2016

Abstract

Introduction

The Health and Retirement Study (HRS) has collected biomarker data over multiple waves. Such data can help improve our understanding of health changes in individuals and the causal pathways related to health. There are, however, technical challenges to using the HRS dried blood spots (DBS) biomarker data due to changes over time in assay protocols, platforms, and laboratories. We provide technical and summary information on biological indicators collected as part of the HRS from 2006 to 2016 that should be helpful to users of the data.

Methods

We describe the opportunities and challenges provided by the HRS DBS data as well as insights provided by the data. The HRS collected DBS from its nationally representative sample of respondents 51 years of age or older from 2006 to 2016. DBS-based biomarkers were collected from half the sample in 2006, 2010, and 2014, and from the other half of the sample in 2008, 2012, and 2016. These DBS specimens were used to assay total and HDL cholesterol, glycosylated hemoglobin, C-reactive protein, and cystatin C from 2006 to 2016, and Interleukin 6 was added in 2014/2016. Samples included approximately 6000 individuals at each wave, and completion rates ranged from 81% to 90%. HRS transformed DBS values into venous blood equivalents to make them more comparable to those of the whole blood-based assays collected in most other studies and to facilitate longitudinal analysis.

Results

Distribution of changes over time by age shows that total cholesterol levels decreased for each age, while HbA1c levels increased. Cystatin C shows a clear age gradient, but a number of other markers do not. Non-Hispanic Black persons and Hispanic respondents have a higher incidence of risk levels of each biomarker except for CRP among non-Hispanic Black older persons.

Conclusion

These public-use DBS data provide analysis opportunities that can be used to improve our understanding of health change with age in both populations and among individuals.

Neuroprotective effects of vitamin D in an Alzheimer’s disease rat model: Improvement of mitochondrial dysfunction via calcium/calmodulin‐dependent protein kinase kinase 2 activation of Sirtuin1 phosphorylation

Neuroprotective effects of vitamin D in an Alzheimer's disease rat model: Improvement of mitochondrial dysfunction via calcium/calmodulin-dependent protein kinase kinase 2 activation of Sirtuin1 phosphorylation

The study investigated the potential role of vitamin D3 (Vit.D) in mitigating cognitive impairment and mitochondrial dysfunction in an Alzheimer's disease (AD) rat model. The results showed that Vit.D improved memory abilities, reduced Aβ aggregation and elevated p-Tau levels, restored normal mitochondrial function, and reduced inflammatory and oxidative stress via CAMKK2-AMPK/SIRT1 pathway upregulation. These findings suggest that Vit.D may have a neuroprotective effect against AD.


Abstract

Mitochondrial dysfunction is an early event in Alzheimer's disease (AD) pathogenesis. To assess the impact of vitamin D3 (Vit.D) on neurogenesis, we investigated its role in mitigating cognitive impairment and mitochondrial dysfunction through calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-mediated phosphorylation of Sirtuin1 (SIRT1) in an aluminum-chloride-D-galactose (AlCl3-D-gal)-induced AD rat model. Rats were distributed into four groups: control, AlCl3 + D-gal (10 + 60 mg/kg, ip), Vit.D (500 IU/kg, po), and AlCl3 + D-gal+Vit.D. Novel object recognition (NOR), Morris Water Maze, and passive avoidance (PA) tests were used to measure memory abilities. The hippocampal tissue was used to assess vitamin D3 receptor (VDR) and peroxisome-proliferator-activated-receptor-γ-coactivator-1α ( PGC-1α) expression by quantitative real-time polymerase chain reaction (qRT-PCR), CAMKK2, p-SIRT1, phosphorylated-AMP-activated protein kinase (p-AMPK), dynamin-related-protein-1 (Drp1), and mitofusin-1 (Mnf1) proteins by western blot and Ca2+ levels, endothelial nitic oxide synthase (eNOS), superoxide dismutase (SOD), amyloid beta (Aβ), and phospho tau (p-Tau) via enzyme-linked immunosorbent assay(ELISA) in addition to histological and ultrastructural examination of rat's brain tissue. Vit.D-attenuated hippocampal injury reversed the cognitive decline and Aβ aggregation, and elevated p-Tau levels in the AlCl3 + D-gal-induced AD rat model. In AlCl3 + D-gal-exposed rats, Vit.D induced VDR expression, normalized Ca2+ levels, elevated CAMKK2, p-AMPK, p-SIRT1, and PGC-1α expression. Vit.D reduced Drp1, induced Mnf1, increased mitochondrial membrane potential, preserved mitochondrial structure, restored normal mitochondrial function, and retained normal eNOS level and SOD activity in AlCl3 + D-gal rats. In conclusion, our findings proved that Vit.D may ameliorate cognitive deficits in AlCl3 + D-gal-induced AD by restoring normal mitochondrial function and reducing inflammatory and oxidative stress via CAMKK2-AMPK/SIRT1 pathway upregulation.

Siderophore specificities of the Pseudomonas aeruginosa TonB‐dependent transporters ChtA and ActA

Siderophore specificities of the Pseudomonas aeruginosa TonB-dependent transporters ChtA and ActA

Iron is a key nutrient for the growth of almost all bacteria. The pathogen Pseudomonas aeruginosa is able to express at least 15 different iron acquisition pathways, each involving a specific outer membrane transporter. Most of these iron-uptake pathways rely on small iron chelators (siderophores) produced by other microorganisms. We identified the outer membrane transporters involved in the uptake of iron via two α-carboxylate siderophores and three mixed α-carboxylate/hydroxamate siderophores.


Iron is an essential nutrient for the survival and virulence of Pseudomonas aeruginosa. The pathogen expresses at least 15 different iron-uptake pathways, the majority involving small iron chelators called siderophores. P. aeruginosa produces two siderophores, but can also use many produced by other microorganisms. This implies that the bacterium expresses appropriate TonB-dependent transporters (TBDTs) at the outer membrane to import the ferric form of each of the siderophores used. Here, we show that the two α-carboxylate-type siderophores rhizoferrin-Fe and staphyloferrin A-Fe are transported into P. aeruginosa cells by the TBDT ActA. Among the mixed α-carboxylate/hydroxamate-type siderophores, we found aerobactin-Fe to be transported by ChtA and schizokinen-Fe and arthrobactin-Fe by ChtA and another unidentified TBDT. Our findings enhance the understanding of the adaptability of P. aeruginosa and hold significant implications for developing novel strategies to combat antibiotic resistance.

Impact of genetic polymorphism on personalized diet and exercise program for steatotic liver disease

Impact of genetic polymorphism on personalized diet and exercise program for steatotic liver disease

Personalized diet and exercise program for steatotic liver disease improved liver function tests, physical findings, glycolipid metabolism markers, and cardiovascular disease risk score. Genetic polymorphism might partially affect treatment efficacy. Further studies should be performed to develop an individualized program for steatotic liver disease, considering genetic polymorphism.


Abstract

Aims

The effects of genetic polymorphism on a personalized diet and exercise program for steatotic liver disease (SLD) are still unclear.

Methods

Participants of this retrospective cohort study were 203 Japanese patients with SLD diagnosed by abdominal ultrasonography. All of them were introduced the personalized diet and exercise treatment. A diet of 25–30 kcal/kg multiplied by ideal body weight (BW) daily and aerobic and resistance exercise (exercise intensity of 4–5 metabolic equivalents daily, respectively) were performed for 6 days. Treatment efficacy was evaluated in terms of the rate of decrease of liver function tests, glycolipid metabolism markers, physical findings, image findings, and cardiovascular disease (CVD) risk score at 6 months compared with baseline. Furthermore, the impact of genetic polymorphism was also investigated.

Results

At 6 months compared with baseline, liver function tests (AST, ALT, γGTP), glycolipid metabolism markers (hemoglobin A1c, triglycerides [TG], low-density lipoprotein cholesterol), physical findings (BW, body mass index), image finding (liver stiffness measurement), and CVD risk score (Suita score) improved significantly. There was no significant difference in treatment efficacy, except for the rates of decrease of TG, according to genotype PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs6834314. The rates of decrease of TG with TM6SF2 CT were significantly higher than those with CC or TT, and the rates of TG with HSD17B13 AA were significantly higher than those with AG by multiple comparisons.

Conclusion

Personalized diet and exercise program for SLD improved liver function tests, physical findings, glycolipid metabolism markers, and CVD risk score. Genetic polymorphism might partially affect treatment efficacy. Further studies should be performed to develop an individualized program for SLD, considering genetic polymorphism.

Significance of the autoantibody assay in predicting the development of immune‐related adverse events in patients receiving atezolizumab plus bevacizumab combination therapy for unresectable hepatocellular carcinoma

Significance of the autoantibody assay in predicting the development of immune-related adverse events in patients receiving atezolizumab plus bevacizumab combination therapy for unresectable hepatocellular carcinoma

It may be difficult to continue atezolizumab plus bevacizumab (AB) combination therapy for unresectable hepatocellular carcinoma (u-HCC) in patients with severe immune-related adverse events (irAEs), and it may lead to deterioration of their general condition. Therefore, it is important to establish a system for predicting the risk of irAE development before starting AB therapy. Several studies have reported that the presence of autoantibodies at baseline may be associated with irAE development in patients with other types of carcinomas. The present study aimed to determine the association between autoantibodies and irAE development in patients receiving AB therapy for u-HCC. Sixteen percent of patients developed irAEs during the observation period. Autoantibodies at baseline was an independent factor associated with irAE development (hazard ratio: 3.7, p = 0.047). Patients with autoantibodies at baseline are at high risk of irAE development and require cautious follow-up.


Abstract

Aim

Atezolizumab plus bevacizumab (AB) combination therapy is the first-line treatment for unresectable hepatocellular carcinoma (u-HCC). The management of immune-related adverse events (irAEs) is an important issue associated with achieving a good therapeutic response in patients receiving AB therapy. However, few studies have reported irAE development in patients receiving AB therapy. This study focused on the association between irAE development and autoantibodies at baseline in patients receiving AB therapy.

Methods

Sixty-one patients receiving AB therapy were enrolled. For autoantibodies, the following antibodies were tested before the start of AB therapy: antinuclear antibodies, rheumatoid factor (RF), anti-thyroglobulin antibodies, thyroid peroxidase antibodies, anti-thyroid stimulating hormone receptor antibodies, and acetylcholine receptor antibodies. A patient was considered to have pre-existing antibodies if any of the listed antibodies were present at baseline.

Results

Ten patients (16%) developed irAEs during the observation period. The irAEs included liver injury, hypothyroidism, adrenal insufficiency, adrenocorticotropic hormone deficiency, and rhabdomyolysis. Patients with irAE (n = 10) were more likely to be positive for any autoantibody (hazard ratio [HR] 3.7, p = 0.047) and RF at baseline (HR 5.4, p = 0.035) and to achieve complete response (HR 5.8, p = 0.027) than those without. The presence of autoantibodies at baseline was an independent factor associated with irAE development.

Conclusion

In the real world, 16% of patients receiving AB therapy for u-HCC developed irAEs. Patients with autoantibodies at baseline are at high risk of developing irAEs and require cautious follow-up.

Survey of the feeding management of giant anteaters (Myrmecophaga tridactyla) and tamanduas (Tamandua tetradactyla) in the EAZA ex‐situ programme

Abstract

Feeding giant anteaters and tamanduas as insectivorous species provides a lot of challenges for zoological institutions. In the last decades an in-house mixture, called ‘Dortmund mixture’, was the most common feed used in giant anteaters and tamanduas in many countries within the European Association of Zoos and Aquaria ex-situ Programme (EEP). Some nutritional problems occurred due to imbalances in the diets. The more recent nutritional recommendations for both species advise an adapted and balanced complete feed formulated for insectivorous species due to different problems arising with an in-house mixed feed as Dortmund mixture. To objectify the present situation a questionnaire was designed and sent out to 78 institutions of the EEPs for giant anteater(s) and tamandua(s). The questionnaire was divided into different sections and asked for data on husbandry, health status, feeding, especially feed composition, feed supplementation and faecal consistency. It was completed by 45 institutions with data for 130 animals, 89 giant anteaters and 41 tamanduas. The data thus represent 54% and 59% of the EEP populations. For both species, a complete feed is mainly utilised. Especially institutions that have integrated anteaters and tamanduas into their facilities during the last 10 and 20 years, use a complete feed. Regarding the in-house mixtures, there are distinct differences, both in composition and amount of each ingredient used. The evaluation of the feeds used for enrichment, for example, shows a clear species difference. While in tamanduas mainly insects are used for this purpose, in giant anteaters it is mainly fruits and avocado. In contrast to the past, many anteaters today are fed an adapted complete feed. Surprisingly, concerning feeding supplements the use of fat-soluble vitamins and combined vitamin–mineral preparations is still common in both species. More effort needs to be put into enforcing current feeding recommendations, especially for the giant anteaters.

The Impact of Non‐obese Metabolic Dysfunction‐associated Fatty Liver Disease on Risk Factors for the Recurrence of Esophageal Squamous Cell Carcinoma Treated with Endoscopic Submucosal Dissection: A Multicenter Study

The Impact of Non-obese Metabolic Dysfunction-associated Fatty Liver Disease on Risk Factors for the Recurrence of Esophageal Squamous Cell Carcinoma Treated with Endoscopic Submucosal Dissection: A Multicenter Study

Metabolic dysfunction-associated fatty liver disease (MAFLD), along with drinking and smoking, is an independent risk factor for the recurrence of esophageal squamous cell carcinoma after endoscopic treatment. Decision tree and random-forest analyses revealed MAFLD as the second most important classifier for recurrence, followed by drinking. Acyclic graphs revealed that MAFLD directly contributes to the recurrence. Moreover, the cumulative incidence of recurrence was significantly higher in the non-obese than that in the obese MAFLD group among abstainers/non-drinkers.


Abstract

Aim

Metabolic dysfunction is a risk factor for esophageal squamous cell carcinoma (ESCC). We investigated the impact of the recently proposed metabolic dysfunction-associated fatty liver disease (MAFLD) and its subtypes on ESCC recurrence after endoscopic treatment.

Methods

This multicenter observational cohort study enrolled consecutive patients newly diagnosed with ESCC after endoscopic treatment. Patients were classified into MAFLD or non-MAFLD groups. The MAFLD group was further classified into non-obese and obese MAFLD groups with a body mass index cutoff value of 25 kg/m2. The impact of MAFLD on the recurrence of ESCC was evaluated using a decision tree algorithm and random forest analysis.

Results

A total of 147 patients (average age, 69 years; male: female, 127:20; observational period, 2.4 years) were enrolled. The 1-, 3-, and 5-year recurrence rates were 2.0%, 21.1%, and 33.7%, respectively. Independent risk factors for the recurrence of ESCC were MAFLD (HR, 2.2812; 95% CI, 1.0497–4.9571; p=0.0373), drinking status, and smoking status. MAFLD was identified as the second most important classifier for recurrence followed by drinking status. The cumulative incidence of ESCC recurrence was higher in the MAFLD group than in the non-MAFLD group. In a sub-analysis, the cumulative incidence of recurrence was significantly higher in the non-obese than in the obese MAFLD group among abstainers/non-drinkers. Directed acyclic graphs revealed that MAFLD directly contributes to ESCC recurrence.

Conclusions

MAFLD was independently and directly associated with ESCC recurrence after endoscopic treatment; a high recurrence rate was observed in patients with non-obese MAFLD. MAFLD may identify patients at high risk for ESCC recurrence.

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