Metabolomics and proteomics insights into subacute ruminal acidosis etiology and inhibition of proliferation of yak rumen epithelial cells in vitro.

BACKGROUND: Untargeted metabolomics and proteomics were employed to investigate the intracellular response of yak rumen epithelial cells (YRECs) to conditions mimicking subacute rumen acidosis (SARA) etiology, including exposure to short-chain fatty acids (SCFA), low pH5.5 (Acid), and lipopolysaccharide (LPS) exposure for 24 h. RESULTS: These treatments significantly altered the cellular morphology of YRECs. Metabolomic analysis identified significant perturbations with SCFA, Acid and LPS treatment affecting 259, 245 and 196 metabolites (VIP > 1, P < 0.05, and fold change (FC) ≥ 1.5 or FC ≤ 0.667). Proteomic analysis revealed that treatment with SCFA, Acid, and LPS resulted in differential expression of 1251, 1396, and 242 proteins, respectively (FC ≥ 1.2 or ≤ 0.83, P < 0.05, FDR < 1%). Treatment with SCFA induced elevated levels of metabolites involved in purine metabolism, glutathione metabolism, and arginine biosynthesis, and dysregulated proteins associated with actin cytoskeleton organization and ribosome pathways. Furthermore, SCFA reduced the number, morphology, and functionality of mitochondria, leading to oxidative damage and inhibition of cell survival. Gene expression analysis revealed a decrease the genes expression of the cytoskeleton and cell cycle, while the genes expression associated with inflammation and autophagy increased (P < 0.05). Acid exposure altered metabolites related to purine metabolism, and affected proteins associated with complement and coagulation cascades and RNA degradation. Acid also leads to mitochondrial dysfunction, alterations in mitochondrial integrity, and reduced ATP generation. It also causes actin filaments to change from filamentous to punctate, affecting cellular cytoskeletal function, and increases inflammation-related molecules, indicating the promotion of inflammatory responses and cellular damage (P < 0.05). LPS treatment induced differential expression of proteins involved in the TNF signaling pathway and cytokine-cytokine receptor interaction, accompanied by alterations in metabolites associated with arachidonic acid metabolism and MAPK signaling (P < 0.05). The inflammatory response and activation of signaling pathways induced by LPS treatment were also confirmed through protein interaction network analysis. The integrated analysis reveals co-enrichment of proteins and metabolites in cellular signaling and metabolic pathways. CONCLUSIONS: In summary, this study contributes to a comprehensive understanding of the detrimental effects of SARA-associated factors on YRECs, elucidating their molecular mechanisms and providing potential therapeutic targets for mitigating SARA.

Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

Low levels of Al stimulate the aboveground growth of Davidia involucrata saplings.

Davidia involucrata is a woody perennial and the only living species in the Genus Davidia. It is native to southern China where it holds cultural and scientific importance. However, D. involucrata is now an endangered species and its natural range includes low pH soils which are increasingly impacted by acid rain, nitrogen deposition and imbalanced nutrient cycling. The combination of these stresses also poses the additional risk of aluminum (Al) toxicity. Since the responses of D. involucrata to low pH and aluminum toxicity have not been investigated previously, a hydroponic experiment was conducted to examine the growth of one year old D. involucrata saplings after 50 d growth in a range of pH and Al conditions. Plant biomass, morphology, antioxidant enzyme activity, mineral concentrations and plant ecological strategy were compared at pH 5.8 and pH 4.0 without added Al (AlCl3) and in 0.1, 0.2 and 0.5 mM Al at pH 4.0. Our results showed that compared with pH 5.8, pH 4.0 (without added Al) not only inhibited root and shoot growth but also limited accumulation of nitrogen (N) and phosphorus (P) in leaves of D. involucrate. However, low Al concentrations (0.1 and 0.2 mM Al) at pH 4.0 partially restored the aboveground growth and leaf N concentrations, suggesting an alleviation of H+ toxicity by low Al concentrations. Compared with low Al concentrations, 0.5 mM Al treatment decreased plant growth and concentrations of N, P, and magnesium (Mg) in the leaves, which demonstrated the toxicity of high Al concentration. The results based on plant ecological strategy showed that D. involucrate decreased the competitiveness and favored its stress tolerance as pH changed from 5.8 to 4.0. Meanwhile, the competitiveness and stress tolerance of D. involucrata increased and decreased at low Al concentrations, respectively, and decreased and increased at high Al concentration, respectively. These trade-offs in ecological strategy were consistent with the responses of growth and antioxidant enzyme activity, reflecting a sensitive adaptation of D. involucrata to acid and Al stresses, which may aid in sustaining population dynamics. These findings are meaningful for understanding the population dynamics of D. involucrata in response to aluminum toxicity in acid soils.

Antibacterial efficacy of mycobacteriophages against virulent Mycobacterium tuberculosis.

Tuberculosis (TB) remains a major global health concern, with drug-resistant strains posing a significant challenge to effective treatment. Bacteriophage (phage) therapy has emerged as a potential alternative to combat antibiotic resistance. In this study, we investigated the efficacy of widely used mycobacteriophages (D29, TM4, DS6A) against Mycobacterium tuberculosis (M. tuberculosis) under pathophysiological conditions associated with TB, such as low pH and hypoxia. We found that even at low multiplicity of infection (MOI), mycobacteriophages effectively infected M. tuberculosis, got rapidly amplified, and lysed M. tuberculosis, demonstrating their potential as therapeutic agents. Furthermore, we observed a novel phage tolerance mechanism with bacteria forming aggregates after several days of phage treatment. These aggregates were enriched with biofilm components and metabolically active bacteria. However, no phage tolerance was observed upon treatment with the three-phage mixture, highlighting the dynamic interplay between phages and bacteria and emphasizing the importance of phage cocktails. We also observed that phages were effective in lysing bacteria even under low pH and low oxygen concentrations as well as antibiotic-resistant bacteria. Our results provide key insights into phage infection of slow-growing bacteria and suggest that mycobacteriophages can effectively eliminate M. tuberculosis in complex pathophysiological environments like hypoxia and acidic pH. These results can aid in developing targeted phage-based therapies to combat antibiotic-resistant mycobacterial infections.

A current insight into Salmonella's inducible acid resistance.

Salmonella is a diverse and ubiquitous group of bacteria and a major zoonotic pathogen implicated in several foodborne disease outbreaks worldwide. With more than 2500 distinct serotypes, this pathogen has evolved to survive in a wide spectrum of environments and across multiple hosts. The primary and most common source of transmission is through contaminated food or water. Although the main sources have been primarily linked to animal-related food products, outbreaks due to the consumption of contaminated plant-related food products have increased in the last few years. The perceived ability of Salmonella to trigger defensive mechanisms following pre-exposure to sublethal acid conditions, namely acid adaptation, has renewed a decade-long attention. The impact of acid adaptation on the subsequent resistance against lethal factors of the same or multiple stresses has been underscored by multiple studies. Α plethora of studies have been published, aiming to outline the factors that- alone or in combination- can impact this phenomenon and to unravel the complex networking mechanisms underlying its induction. This review aims to provide a current and updated insight into the factors and mechanisms that rule this phenomenon.

Molecular basis for the different PCV2 susceptibility of T-lymphoblasts in Landrace and Piétrain pigs.

Clinically, Landrace pigs are more susceptible to porcine circovirus-associated diseases (PCVADs) than Piétrain pigs. We previously found that porcine circovirus type 2 (PCV2) can infect T-lymphoblasts. The present study examined the replication kinetics of six PCV2 strains in the lymphoblasts of Landrace and Piétrain pigs. The results showed that T-lymphoblasts from Landrace pigs are much more susceptible to PCV2 infection than those from Piétrain pigs. In addition, PCV2 replication was strain-dependent. PCV2 binding to T-lymphoblasts was partially mediated by chondroitin sulfate (CS) and dermatan sulfate (DS). Phosphacan, an effective internalization mediator in monocytes that contains several CS chains, was also demonstrated to be involved in PCV2 internalization. Viral binding and internalization were not different between the two breeds, however, the subsequent step, the disassembly was. Although inhibition of serine proteases blocked PCV2 replication in both Landrace and Piétrain pigs, this only occurred at a neutral pH in Piétrain pigs, whereas this occurred also at a low pH in Landrace. This suggested that more proteases can cleave PCV2 in Landrace lymphoblasts than in Piétrain lymphoblasts, explaining the better replication. Through co-localization studies of viral particles with endo-lysosomal markers, and quantitative analysis of organelle sizes during viral internalization, it was observed that PCV2 may exhibit a higher propensity for viral escape from late endosomes in Landrace pigs (smaller) compared to Piétrain pigs. These results provide new understandings of the different PCV2 susceptibility in Landrace and Piétrain pigs.

Transcriptional response of Saccharomyces cerevisiae to lactic acid enantiomers.

The model yeast, Saccharomyces cerevisiae, is a popular object for both fundamental and applied research, including the development of biosensors and industrial production of pharmaceutical compounds. However, despite multiple studies exploring S. cerevisiae transcriptional response to various substances, this response is unknown for some substances produced in yeast, such as D-lactic acid (DLA). Here, we explore the transcriptional response of the BY4742 strain to a wide range of DLA concentrations (from 0.05 to 45 mM), and compare it to the response to 45 mM L-lactic acid (LLA). We recorded a response to 5 and 45 mM DLA (125 and 113 differentially expressed genes (DEGs), respectively; > 50% shared) and a less pronounced response to 45 mM LLA (63 DEGs; > 30% shared with at least one DLA treatment). Our data did not reveal natural yeast promoters quantitatively sensing DLA but provide the first description of the transcriptome-wide response to DLA and enrich our understanding of the LLA response. Some DLA-activated genes were indeed related to lactate metabolism, as well as iron uptake and cell wall structure. Additional analyses showed that at least some of these genes were activated only by acidic form of DLA but not its salt, revealing the role of pH. The list of LLA-responsive genes was similar to those published previously and also included iron uptake and cell wall genes, as well as genes responding to other weak acids. These data might be instrumental for optimization of lactate production in yeast and yeast co-cultivation with lactic acid bacteria. KEY POINTS: • We present the first dataset on yeast transcriptional response to DLA. • Differential gene expression was correlated with yeast growth inhibition. • The transcriptome response to DLA was richer in comparison to LLA.

pH inactivation of SARS-CoV-2 and SARS-CoV in virus spiked protein A eluates from a mAb purification process.

Biopharmaceutical manufacturing processes may include a low pH treatment step as a means of inactivating enveloped viruses. Small scale virus clearance studies are routinely performed using model enveloped viruses such as murine leukemia virus to assess inactivation at the pH range used in the downstream manufacturing process. Further, as a means of bioburden reduction, chromatography resins may be cleaned and stored using sodium hydroxide and this can also inactivate viruses. The susceptibility of SARS-CoV-2 and SARS-CoV to low pH conditions using protein A eluate derived material from a monoclonal antibody production process as well as high pH cleaning conditions was addressed. SARS-CoV-2 was effectively inactivated at pH 3.0, moderately inactivated at pH 3.4, but not inactivated at pH 3.8. Low pH was less effective at inactivating SARS-CoV. Both viruses were inactivated at a high pH of ca.13.4. These studies provide important information regarding the effectiveness of viral clearance and inactivation steps of novel coronaviruses when compared to other enveloped viruses.

Successful operation of nitrifying granules at low pH in a continuous-flow reactor: Nitrification performance, granule stability, and microbial community.

Acidic nitrification, as a novel process for treating wastewater without sufficient alkalinity, has received increasing attention over the years. In this study, a continuous-flow reactor with aerobic granular sludge was successful operated at low pH (<6.5) performing high-rate acidic nitrification. Volumetric ammonium oxidation rate of 0.4-1.2 kg/(m3·d) were achieved with the specific biomass activities of 5.8-13.9 mg N/(gVSS·h). Stable partial nitritation with nitrite accumulation efficiency over 85% could be maintained at pH above 6 with the aid of residual ammonium, whereas the nitrite accumulation disappeared when pH was below 6. Interestingly, the granule morphology significantly improved during the acidic operation. The increased secretion of extracellular polymeric substances (especially polysaccharides) suggested a self-protective behavior of microbes in the aerobic granules against acidic stress. 16S rRNA gene sequencing analyses indicated that Candidatus Nitrospira defluvii was always the dominant nitrite-oxidizing bacteria, while the dominant ammonia-oxidizing bacteria shifted from Nitrosomonas europaea to Nitrosomonas mobilis. This study, for the first time, demonstrated the improved stability of aerobic granules under acidic conditions, and also highlighted aerobic granules as a useful solution to achieve high-rate acidic nitrification.

Chemical Evolution of Rhinovirus Identifies Capsid-Destabilizing Mutations Driving Low-pH-Independent Genome Uncoating.

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

Improving the cytotoxicity of immunotoxins by reducing the affinity of the antibody in acidic pH.

BACKGROUND: Immunotoxins are antibody-toxin conjugates that bind to surface antigens and exert effective cytotoxic activity after internalization into tumor cells. Immunotoxins exhibit effective cytotoxicity and have been approved by the FDA to treat multiple hematological malignancies, such as hairy cell leukemia and cutaneous T-cell lymphoma. However, most of the internalized immunotoxin is degraded in lysosomes, and only approximately 5% of free toxin escapes into the cytosol to exert cytotoxicity. Many studies have improved immunotoxins by engineering the toxin fragment to reduce immunogenicity or increase stability, but how the antibody fragment contributes to the activity of immunotoxins has not been well demonstrated. METHODS: In the current study, we used 32A9 and 42A1, two anti-GPC3 antibodies with similar antigen-binding capabilities and internalization rates, to construct scFv-mPE24 immunotoxins and evaluated their in vitro and in vivo antitumor activities. Next, the antigen-binding capacity, trafficking, intracellular protein stability and release of free toxin of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 were compared to elucidate their different antitumor activities. Furthermore, we used a lysosome inhibitor to evaluate the degradation behavior of 32A9 scFv-mPE24 and 42A1 scFv-mPE24. Finally, the antigen-binding patterns of 32A9 and 42A1 were compared under neutral and acidic pH conditions. RESULTS: Although 32A9 and 42A1 had similar antigen binding capacities and internalization rates, 32A9 scFv-mPE24 had superior antitumor activity compared to 42A1 scFv-mPE24. We found that 32A9 scFv-mPE24 exhibited faster degradation and drove efficient free toxin release compared to 42A1 scFv-mPE24. These phenomena were determined by the different degradation behaviors of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 in lysosomes. Moreover, 32A9 was sensitive to the low-pH environment, which made the 32A9 conjugate easily lose antigen binding and undergo degradation in lysosomes, and the free toxin was then efficiently produced to exert cytotoxicity, whereas 42A1 was resistant to the acidic environment, which kept the 42A1 conjugate relatively stable in lysosomes and delayed the release of free toxin. CONCLUSIONS: These results showed that a low pH-sensitive antibody-based immunotoxin degraded faster in lysosomes, caused effective free toxin release, and led to improved cytotoxicity compared to an immunotoxin based on a normal antibody. Our findings suggested that a low pH-sensitive antibody might have an advantage in the design of immunotoxins and other lysosomal degradation-dependent antibody conjugate drugs.

Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH.

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date.

Ocean acidification alters foraging behaviour in Dungeness crab through impairment of the olfactory pathway.

Crustacean olfaction is fundamental to most aspects of living and communicating in aquatic environments and more broadly, for individual- and population-level success. Accelerated ocean acidification from elevated CO2 threatens the ability of crabs to detect and respond to important olfactory-related cues. Here, we demonstrate that the ecologically and economically important Dungeness crab (Metacarcinus magister) exhibits reduced olfactory-related antennular flicking responses to a food cue when exposed to near-future CO2 levels, adding to the growing body of evidence of impaired crab behaviour. Underlying this altered behaviour, we find that crabs have lower olfactory nerve sensitivities (twofold reduction in antennular nerve activity) in response to a food cue when exposed to elevated CO2 . This suggests that near-future CO2 levels will impact the threshold of detection of food by crabs. We also show that lower olfactory nerve sensitivity in elevated CO2 is accompanied by a decrease in the olfactory sensory neuron (OSN) expression of a principal chemosensory receptor protein, ionotropic receptor 25a (IR25a) which is fundamental for odorant coding and olfactory signalling cascades. The OSNs also exhibit morphological changes in the form of decreased surface areas of their somata. This study provides the first evidence of the effects of high CO2 levels at multiple levels of biological organization in marine crabs, linking physiological and cellular changes with whole animal behavioural responses.

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification.

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log10 reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65-3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.

Viral clearance in end-to-end integrated continuous process for mAb purification: Total flow-through integrated polishing on two columns connected to virus filtration.

There are few reports of the adoption of continuous processes in bioproduction, particularly the implementation of end-to-end continuous or integrated processes, due to difficulties such as feed adjustment and incorporating virus filtration. Here, we propose an end-to-end integrated continuous process for a monoclonal antibody (mAb) with three integrated process segments: upstream production processes with pool-less direct connection, pooled low pH virus inactivation with pH control and a total flow-through integrated polishing process in which two columns were directly connected with a virus filter. The pooled virus inactivation step defines the batch, and high impurities reduction and mAb recovery were achieved for batches conducted in succession. Viral clearance tests also confirmed robust virus reduction for the flow-through two-column chromatography and the virus filtration steps. Additionally, viral clearance tests with two different hollow fiber virus filters operated at flux ranging from 1.5 to 40 LMH (liters per effective surface area of filter in square meters per hour) confirmed robust virus reduction over these ranges. Complete clearance with virus logarithmic reduction value ≥4 was achieved even with a process pause at the lowest flux. The end-to-end integrated continuous process proposed in this study is amenable to production processes, and the investigated virus filters have excellent applicability to continuous processes conducted at constant flux.

Raman Spectroscopic Analysis of Highly-Concentrated Antibodies under the Acid-Treated Conditions.

PURPOSE: Antibody drugs are usually formulated as highly-concentrated solutions, which would easily generate aggregates, resulting in loss of efficacy. Although low pH increases the colloidal dispersion of antibodies, acid denaturation can be an issue. Therefore, knowing the physical properties at low pH under high concentration conditions is important. METHODS: Raman spectroscopy was used to investigate pH-induced conformational changes of antibodies at 50 mg/ml. Experiments in pH 3 to 7 were performed for human serum IgG and recombinant rituximab. RESULTS: We detected the evident changes at pH 3 in Tyr and Trp bands, which are the sensitive markers of intermolecular interactions. Thermal transition analysis over the pH range demonstrated that the thermal transition temperature (Tm) was highest at pH 3. Acid-treated and neutralized one showed higher Tm than that of pH 7, indicating that their extent of intermolecular interactions correlated with the Tm values. Onset temperature was clearly different between concentrated and diluted samples. Colloidal analyses confirmed the findings of the Raman analysis. CONCLUSION: Our studies demonstrated the positive correlation between Raman analysis and colloidal information, validating as a method for evaluating antibody conformation associated with aggregation propensities.

Acid waters in tank bromeliads: Causes and potential consequences.

PREMISE: The consequences of acidity for plant performance are profound, yet the prevalence and causes of low pH in bromeliad tank water are unknown despite its functional relevance to key members of many neotropical plant communities. METHODS: We investigated tank water pH for eight bromeliad species in the field and for the widely occurring Guzmania monostachia in varying light. We compared pH changes over time between plant and artificial tanks containing a solution combined from several plants. Aquaporin transcripts were measured for field plants at two levels of pH. We investigated relationships between pH, leaf hydraulic conductance, and CO2 concentration in greenhouse plants and tested proton pump activity using a stimulator and inhibitor. RESULTS: Mean tank water pH for the eight species was 4.7 ± 0.06 and was lower for G. monostachia in higher light. The pH of the solution in artificial tanks, unlike in plants, did not decrease over time. Aquaporin transcription was higher for plants with lower pH, but leaf hydraulic conductance did not differ, suggesting that the pH did not influence water uptake. Tank pH and CO2 concentration were inversely related. Fusicoccin enhanced a decrease in tank pH, whereas orthovanadate did not. CONCLUSIONS: Guzmania monostachia acidified its tank water via leaf proton pumps, which appeared responsive to light. Low pH increased aquaporin transcripts but did not influence leaf hydraulic conductance, hence may be more relevant to nutrient uptake.

Understanding the Streptomyces albulus response to low-pH stress at the interface of physiology and transcriptomics.

Streptomyces albulus is a well-established cell factory for ε-poly-L-lysine (ε-PL) production. It has been reported that ε-PL biosynthesis is strictly regulated by pH and that ε-PL can accumulate at approximately pH 4.0, which is outside of the general pH range for natural product production by Streptomyces species. However, how S. albulus responds to low pH is not clear. In this study, we attempted to explore the response of S. albulus to low-pH stress at the physiological and global gene transcription levels. At the physiological level, S. albulus maintained intracellular pH homeostasis at ~pH 7.5, increased the unsaturated fatty acid ratio, extended the fatty acid chain length, enhanced ATP accumulation, increased H+-ATPase activity, and accumulated the basic amino acids L-lysine and L-arginine. At the global gene transcription level, carbohydrate metabolism, oxidative phosphorylation, macromolecule protection and repair, and the acid tolerance system were found to be involved in combating low-pH stress. Finally, we preliminarily evaluated the effect of the acid tolerance system and cell membrane fatty acid synthesis on low-pH tolerance via gene manipulation. This work provides new insight into the adaptation mechanism of Streptomyces to low-pH stress and a new opportunity for constructing robust S. albulus strains for ε-PL production. KEY POINTS: • S. albulus consistently remained pH i at ~7.4 regardless of the environmental pH. • S. albulus combats low-pH stress by modulating lipid composition of cell membrane. • Overexpression of cfa in S. albulus could improve low-pH tolerance and ɛ-PL titer.

Overexpression of ZmSTOP1-A Enhances Aluminum Tolerance in Arabidopsis by Stimulating Organic Acid Secretion and Reactive Oxygen Species Scavenging.

Aluminum (Al) toxicity and low pH are major factors limiting plant growth in acidic soils. Sensitive to Proton Rhizotoxicity 1 (STOP1) transcription factors respond to these stresses by regulating the expression of multiple Al- or low pH-responsive genes. ZmSTOP1-A, a STOP1-like protein from maize (Zea mays), was localized to the nucleus and showed transactivation activity. ZmSTOP1-A was expressed moderately in both roots and shoots of maize seedlings, but was not induced by Al stress or low pH. Overexpression of ZmSTOP1-A in Arabidopsis Atstop1 mutant partially restored Al tolerance and improved low pH tolerance with respect to root growth. Regarding Al tolerance, ZmSTOP1-A/Atstop1 plants showed clear upregulation of organic acid transporter genes, leading to increased organic acid secretion and reduced Al accumulation in roots. In addition, the antioxidant enzyme activity in roots and shoots of ZmSTOP1-A/Atstop1 plants was significantly enhanced, ultimately alleviating Al toxicity via scavenging reactive oxygen species. Similarly, ZmSTOP1-A could directly activate ZmMATE1 expression in maize, positively correlated with the number of Al-responsive GGNVS cis-elements in the ZmMATE1 promoter. Our results reveal that ZmSTOP1-A is an important transcription factor conferring Al tolerance by enhancing organic acid secretion and reactive oxygen species scavenging in Arabidopsis.

Organic Additive-derived Films on Cu Electrodes Promote Electrochemical CO2 Reduction to C2+ Products Under Strongly Acidic Conditions.

Electrochemical CO2 reduction (CO2 R) at low pH is desired for high CO2 utilization; the competing hydrogen evolution reaction (HER) remains a challenge. High alkali cation concentration at a high operating current density has recently been used to promote electrochemical CO2 R at low pH. Herein we report an alternative approach to selective CO2 R (>70 % Faradaic efficiency for C2+ products, FEC2+ ) at low pH (pH 2; H3 PO4 /KH2 PO4 ) and low potassium concentration ([K+ ]=0.1 M) using organic film-modified polycrystalline copper (Modified-Cu). Such an electrode effectively mitigates HER due to attenuated proton transport. Modified-Cu still achieves high FEC2+ (45 % with Cu foil /55 % with Cu GDE) under 1.0 M H3 PO4 (pH≈1) at low [K+ ] (0.1 M), even at low operating current, conditions where HER can otherwise dominate.