Molecular Biology Tools Research Articles

Fast, Simultaneous Tagging and Mutagenesis of Genes on Bacterial Chromosomes.

Fluorescence microscopy has become a powerful tool in molecular cell biology. Visualizing specific proteins in bacterial cells requires labeling with fluorescent or fluorogenic tags, preferentially at the native chromosomal locus to preserve expression dynamics associated with the genomic environment. Exploring protein function calls for targeted mutagenesis and observation of differential phenotypes. In the model bacterium Escherichia coli, protocols for tagging genes and performing targeted mutagenesis currently involve multiple steps. Here, we present an approach capable of simultaneous tagging and mutagenesis of essential and nonessential genes in a single step. We require only the insertion of a stretch of the target gene into an auxiliary plasmid together with the tag. Recombineering-based exchange with the native locus is then carried out, where the desired mutation is introduced during amplification with homology-bearing primers. Using this approach, multiple tagged mutants per gene can be derived quickly.

Mechanisms of Cadmium Accumulation in Plants

Cadmium is a non-essential trace metal, which is highly toxic to nearly all living organisms. Soil pollution causes Cd contamination of crops, thereby rendering plant products responsible for the chronic low level Cd over-exposure of numerous populations in the world. For this reason, Cd accumulation in plants has been studied for about five decades now. The research first focused on the relationships between plant and soil Cd levels, on the factors of the metal availability in soil, as well as the root uptake processes. Cd distribution in plant organs was also investigated, first using a macroscopic and eco-physiological approach, and then with the help of molecular biology tools, at both tissue and cell scales. Cadmium has no biological function and hijacks the transport pathways of micronutrients such as Fe, Mn, or Zn, in order to enter the plant through the roots and be distributed to all its organs. The study of the genes that control the influx and efflux of the Cd2+ ion in the cytosol, vacuoles, and vascular tissues has significantly contributed to the understanding of the metal root uptake and of its transfer to the aerial parts. However, the mechanisms responsible for its distribution to the different above-ground tissues and specially to fruits and seeds have yet to be clarified. This review summarizes current knowledge in order to present a detailed overview of Cd transport and storage, from the rhizosphere to the different organs and tissues of the plant.

Synthetic Biology Bicistronic Designs Support Gene Expression Equally Well in vitro and in vivo

Synthetic biology integrates molecular biology tools and an engineering mindset to address challenges in medicine, agriculture, bioremediation, and biomanufacturing. A persistent problem in synthetic biology has been designing genetic circuits that produce predictable levels of protein. In 2013, Mutalik and colleagues developed bicistronic designs (BCDs) that make protein production more predicable in bacterial cells (in vivo). With the growing interest in producing proteins outside of cells (in vitro), we wanted to know if BCDs would work as predictably in cell-free protein synthesis (CFPS) as they do in E. coli cells. We tested 20 BCDs in CFPS and found they performed very similarly in vitro and in vivo. As a step toward developing methods for protein production in artificial cells, we also tested 3 BCDs inside nanoliter-scaled microfluidic droplets. The BCDs worked well in the microfluidic droplets, but their relative protein production levels were not as predictable as expected. These results suggest that the conditions under which gene expression happens in droplets result in a different relationship between genetic control elements such as BCDs and protein production than exists in batch CFPS or in cells. KEYWORDS: Bicistronic Design; Synthetic Biology; Cell-Free Protein Synthesis; Microfluidics

The CCCryo Culture Collection of Cryophilic Algae as a valuable bioresource for algal biodiversity and for novel, industrially marketable metabolites

ABSTRACT The CCCryo Culture Collection of Cryophilic Algae is a diverse bioresource of mainly cold-adapted microalgae from polar environments. It currently comprises 518 strains of 178 species in 101 genera. Most strains were isolated from field samples collected during seven polar expeditions, mainly to Svalbard (Norway) and Antarctica. Almost 90% of the strains are cryophilic, with 33% of all strains being psychrophilic (obligate cryophilic) representing the “true” snow algae and 56% being psychrotrophic (non-obligate cryophilic), comprising other snow and permafrost algae from cold habitats. There are also some cultures of mosses, cyanobacteria, fungi and bacteria from cold or thermal environments. These strains are publicly available and serve as a rare bioresource for fundamental studies as well as for the development of commercial products. Basic studies address taxonomic and phylogenetic as well as physiological questions and genome and transcriptome research, whereas applied studies mostly address the capabilities of cryophilic algae to produce commercially interesting products for markets like food, feed and supplements, cosmetics, tools for molecular biology and diagnostics or food processing. Examples of strains studied for the production of valuable polyunsaturated omega-3 fatty acids like EPA, secondary carotenoids like astaxanthin, ice-structuring proteins, cold-active enzymes, UV-absorbing mycosporine-like amino acids and extracellular polymeric substances as form stabilizers are given and discussed in the context of customers’ acceptance, legal regulations for specific markets, technical feasibility of an industrial production process, potential profit and chances for the successful development of a commercial product.

Biopolymer/plasmid DNA microspheres as tracers for multiplexed hydrological investigation

Pollutants being discharged into water body from unknown sources are one of the major threats to environmental sustainability and human health. Herein a plasmid DNA (pDNA)-based tracing system for multiplexed detection of potential hydrological processes of pollutants is developed. pDNA is incorporated with magnetic nanoparticles (MNPs) and then the pDNA-MNPs nanocomposites are encapsulated inside polylactic acid (PLA) microspheres as pDNA tracers. pDNA, produced in large quantities by engineering bacteria (E. coli), served as the “marker” which can be arbitrarily designed and specifically identified through well-established molecular biology tools including enzyme. MNPs are employed to facilitate the collection and separation of tracers in diluted water body. PLA microspheres provide effective protections for pDNA against unpredictable influences in environment such as high temperature, UV radiation, and deoxyribonuclease degradation. In addition, pDNA tracers containing MNPs are buoyant and concentrated at the surface of the water, facilitating tracer collection with an external magnetic device. pDNA extracted from tracers is specifically detected via quantitative Polymerase Chain Reaction (qPCR) with extreme sensitivity. Multiplexed detection is also achieved both in a laboratory test and in a simulated-stream test. Moreover, the preliminary application strategies of pDNA tracer system in different scales of a real-world river have been envisioned. The advantages of pDNA-based tracing system include: massive and cost-effective production of pDNA, high stability in harsh environment, environmental friendly, ultrasensitive, and allowing for multiplexed detection.

Double-Membrane Vesicles as Platforms for Viral Replication.

Viruses, as obligate intracellular parasites, exploit cellular pathways and resources in a variety of fascinating ways. A striking example of this is the remodelling of intracellular membranes into specialized structures that support the replication of positive-sense ssRNA (+RNA) viruses infecting eukaryotes. These distinct forms of virus-induced structures include double-membrane vesicles (DMVs), found during viral infections as diverse and notorious as those of coronaviruses, enteroviruses, noroviruses, or hepatitis C virus. Our understanding of these DMVs has evolved over the past 15 years thanks to advances in imaging techniques and modern molecular biology tools. In this article, we review contemporary understanding of the biogenesis, structure, and function of virus-induced DMVs as well as the open questions posed by these intriguing structures.

Know Thine Enemy: Viral Genome Sequencing in Outbreaks

CONTAINING a viral outbreak with public health measures firstly requires identification of the causative virus, followed by more detailed understanding of viral features. Genomic sequencing provides exhaustive insight into viral features that may help predict outbreak behaviours, assist in diagnosis and tracking, and shape treatment and vaccination strategies. When coupled with epidemiologic study of outbreak data, viral genomic sequencing can be used to direct public health measures and increase the speed of understanding compared to epidemiology alone. Community spread of cases can be used to guide mathematic models and contact tracing of viral outbreaks for public health response. However, epidemiologic data alone better suits responses to low-prevalence and less-widespread outbreaks. Where pathogens have a longer latency period or spread affects rural and remote communities, features of the virus itself must be considered in determining the response. Genotypic and phenotypic characteristics, identified using molecular biology tools, can clarify the type and strain of a virus responsible for an outbreak, and inform and improve case diagnosis, treatment options, and vaccine development, as well as improve tracing accuracy.1

Production and Purification of Artificial Circular RNA Sponges for Application in Molecular Biology and Medicine.

Characterized by their covalently closed structure and thus an elevated stability compared to linear RNA molecules, circular RNAs (circRNAs) form a novel class of mainly non-coding RNAs. Although the biological functions of naturally occurring circRNAs are largely unknown, they were reported to act as molecular sponges, sequestering microRNAs (miRNAs), resulting in a de-repression of target mRNAs. Taking these characteristics of naturally occurring circRNAs into account, artificial circRNAs could be a potential tool in molecular biology and medicine. Using the Hepatitis C virus (HCV) as a model system, this application of artificial circular RNAs was demonstrated. The virus requires cellular miRNA miR-122 for its life cycle, and circRNAs specifically engineered to efficiently sequester this miRNA impacted viral propagation. Since in this context the production of engineered circRNA remains the limiting factor, we present a method to produce and efficiently purify artificial circRNA sponges (ciRS) in vitro. In this protocol we provide insights into a small-scale and large-scale production technique of artificial circular RNA sponges relying on in vitro transcription and RNA ligation.

Plasmonic Nanosensors for the Label-Free Imaging of Dynamic Protein Patterns.

We introduce a new approach to monitor the dynamics and spatial patterns of biological molecular assemblies. Our molecular imaging method relies on plasmonic gold nanoparticles as point-like detectors and requires no labeling of the molecules. We show spatial resolution of up to 5 μm and 30 ms temporal resolution, which is comparable to wide-field fluorescence microscopy, while requiring only readily available gold nanoparticles and a dark-field optical microscope. We demonstrate the method on MinDE proteins attaching to and detaching from lipid membranes of different composition for 24 h. We foresee our new imaging method as an indispensable tool in advanced molecular biology and biophysics laboratories around the world.

Aerobic and oxygen-limited naphthalene-amended enrichments induced the dominance of Pseudomonas spp. from a groundwater bacterial biofilm

In this study, we aimed at determining the impact of naphthalene and different oxygen levels on a biofilm bacterial community originated from a petroleum hydrocarbon–contaminated groundwater. By using cultivation-dependent and cultivation-independent approaches, the enrichment, identification, and isolation of aerobic and oxygen-limited naphthalene degraders was possible. Results indicated that, regardless of the oxygenation conditions, Pseudomonas spp. became the most dominant in the naphthalene-amended selective enrichment cultures. Under low-oxygen conditions, P. veronii/P. extremaustralis lineage affiliating bacteria, and under full aerobic conditions P. laurentiana–related isolates were most probably capable of naphthalene biodegradation. A molecular biological tool has been developed for the detection of naphthalene 1,2-dioxygenase-related 2Fe-2S reductase genes of Gram-negative bacteria. The newly developed COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP-PCR) technique may be used in the monitoring of the natural attenuation capacity of PAH-contaminated sites. A bacterial strain collection with prolific biofilm-producing and effective naphthalene-degrading organisms was established. The obtained strain collection may be applicable in the future for the development of biofilm-based bioremediation systems for the elimination of PAHs from groundwater (e.g., biofilm-based biobarriers).

Use of the HRM Method in Quick Identification of FecXO Mutation in Highly Prolific Olkuska Sheep.

Simple SummaryAccording to the available literature, high prolificacy in sheep breeds could be caused by differences in many genes (polygenic trait) or as in some sheep breeds to a difference in one gene such as BMP-15. This paper describes the use of the High-Resolution Melting method in quick identification of the known mutation in the BMP-15 gene, which affects high prolificacy in the Olkuska breed of sheep.Olkuska is a highly prolific sheep breed in Poland. Thanks to earlier identification of the genetic basis of its prolificacy, a mutation in the BMP-15 gene, we can use molecular biology tools to identify this causative mutation affecting prolificacy. In our research, we used the High-Resolution Melting (HRM) and Sanger sequencing methods to identify the genotypes of the studied animals. The result obtained by the HRM method is identical to those obtained by the sequencing method, which confirms the effectiveness of the HRM method and the possibility of quick and cheap identification of individuals with a FecXO mutation.

A computational subtractive genome analysis for the characterization of novel drug targets in Klebsiella pneumonia strain PittNDM01

The emergence of carbapenem-resistant Klebsiella Pneumoniae had been reported previously, which needs rapid attention. Currently, Pittsburgh University Hospital reported a new strain of carbapenem-resistant Klebsiella pneumoniae that was co-producing OXA-232 and NDM-1 named as PittNDM01. This strain is resistant to almost all beta-lactam antibiotics such as Carbapenem as well as to fluoroquinolones and aminoglycosides. Globally, failure to the wide-spread pathogenic strains had been observed due to the increased and antibiotic resistance, which leads to less antimicrobial drug efficacy. Since last decades, computational genomic approaches have been introduced to fight against resistant pathogens, which is an advanced approach for novel drug targets investigation. The current study emphasizes the utilization of the available genomic and proteomic data of Klebsiella pneumoniae PittNDM01 for the identification of novel drug targets for future drug developments. Comparative genomic analysis and molecular biological tools were applied, results in observing 582 non-human homologous-essential proteins of Klebsiella pneumoniae. Among the total 582 proteins, 66 were closely related to the pathogen-specific pathway. Out of all 66-targeted proteins, ten non-homologous essential proteins were found to have druggability potential. The subcellular localization of these proteins revealed; 6 proteins in the cytoplasm, 2 in the inner membrane, and one each in periplasmic space and outer membrane. All the above 10 proteins were compared to the proteins sequences of gut flora to eliminate the homologous proteins. In total, 6-novel non-human and non-gut flora essential drug targets of Klebsiella pneumoniae PittNDM01 strain were identified. Further, the 3D structures of the identified drug target proteins were developed, and protein-protein interaction network analysis was performed to know the functional annotation of the desire proteins. Therefore, these non-homologous essential targets ensure the survival of the pathogen and hence can be targeted for drug discovery.

Microbial community functional structure in an aerobic biofilm reactor: Impact of streptomycin and recovery

Antibiotics can affect microbial community structure and promote antibiotic resistance. However, the course of microbial community recovery in wastewater treatment systems after antibiotic disturbance remains unclear. Herein, multiple molecular biology tools, including 16S amplicon sequencing, GeoChip 5.0, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing, were used to investigate the year-long (352 d) recovery of the microbial community functional structure in an aerobic biofilm reactor. Nitrification was completely inhibited under 50 mg/L of streptomycin spiking (STM_50) due to the significant reduction of ammonia-oxidizing bacteria, but recovered to original pre-disturbance levels after streptomycin removal, indicating the high resilience of ammonia-oxidizing bacteria. Bacterial community richness and diversity decreased significantly under STM_50 (p < 0.05), but recovered to levels similar to those observed before disturbance after 352 d. In contrast, bacterial composition did not recover to the original structure. The carbon degradation and nitrogen cycling functional community significantly changed after recovery compared to that observed pre-disturbance (p < 0.05), thus indicating functional redundancy. Additionally, levels of aminoglycoside and total antibiotic resistance genes under STM_50 (relative abundance, 0.33 and 0.80, respectively) and after one year of recovery (0.12 and 0.29, respectively) were higher than the levels detected pre-disturbance (0.04 and 0.24, respectively). This study provides an overall depiction of the recovery of the microbial community functional structure after antibiotic exposure. Our findings give notice that recovery caused by antibiotic disturbance in the water environment should be taken more seriously, and that engineering control strategies should be implemented to prevent the antibiotic pollution of wastewater.

Development of molecular biological tools for the rapid determination of antibiotic susceptibility of Mycoplasma hyopneumoniae isolates

Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, a contagious respiratory disease, causing significant economic losses worldwide. Antibiotic treatment is commonly utilised in the pig industry to control M. hyopneumoniae infection. Since the conventional antibiotic susceptibility test is time-consuming, taking up to weeks’ period, antibiotics are usually empirically chosen.Certain single nucleotide polymorphisms in the parC (C239A/T, G250A) and gyrA (G242C, C247 T, A260 G) genes show correlation with decreased fluoroquinolone susceptibility by the change of the target site. Furthermore, the nucleotide alteration A2059 G in the 23S rRNA sequence correlates with significantly decreased macrolide and lincosamide susceptibility of M. hyopneumoniae. Mismatch amplification mutation assays (MAMA) and high resolution melt (HRM) analysis, capable to detect the mentioned resistance markers, were developed in the present study, in order to provide susceptibility data in a considerably shorter time than the conventional methods. The results of the MAMA and HRM assays were congruent with the results of the conventional antibiotic susceptibility method of the tested M. hyopneumoniae field isolates. The sensitivity of the MAMAs was 103-104 copy numbers, while that of the HRM assay was 105-106 copy numbers.To the best of our knowledge this was the first time that MAMA and HRM assays were developed for the rapid detection of decreased fluoroquinolone, macrolide or lincosamide susceptibility in M. hyopneumoniae strains.

Unravelling the menace: detection of antimicrobial resistance in aquaculture.

One of the major problems to be addressed in aquaculture is the prominence of antimicrobial resistance (AMR). The occurrence of bacterial infections in cultured fishes promotes the continuous use of antibiotics in aquaculture, which results in the selection of proliferated antibiotic-resistant bacteria and increases the possibility of transfer to the whole environment through horizontal gene transfer. Hence, the accurate cultivation-dependent and cultivation-independent detection methods are very much crucial for the immediate and proper management of this menace. Antimicrobial resistance determinants carrying mobile genetic transfer elements such as transposons, plasmids, integrons and gene cassettes need to be specifically analysed through molecular detection techniques. The susceptibility of microbes to antibiotics should be tested at regular intervals along with various biochemical assays and conjugation studies so as to determine the extent of spread of AMR. Advanced omic-based and bioinformatic tools can also be incorporated for understanding of genetic diversity. The present review focuses on different detection methods to unearth the complexity of AMR in aquaculture. This monitoring helps the authorities to curb the use of antibiotics, commencement of appropriate management measures and adequate substitute strategies in aquaculture. The long battle of AMR could be overcome by the sincere implementation of One Health approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of antibiotics and increased antimicrobial resistance (AMR) are of major concerns in aquaculture industry. This could result in global health risks through direct consumption of cultured fishes and dissemination of AMR to natural environment through horizontal gene transfer. Hence, timely detection of the antimicrobial-resistant pathogens and continuous monitoring programmes are inevitable. Advanced microbiological, molecular biological and omic-based tools can unravel the menace to a great extent. This will help the authorities to curb the use of antibiotics and implement appropriate management measures to overcome the threat.

Pandemic SARS Coronavirus-2 Infections in Humans-COVID-19

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) first broke out in Wuhan (China) and subsequently spread worldwide. Coronaviruses (CoVs) primarily cause zoonotic infections in birds and mammals however, in the last few decades have shown to be capable of infecting humans as well. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and more recently, Middle-East respiratory syndrome, (MERS) has demonstrated the lethality of CoVs when they cross the species barrier and infect humans. Coronavirus (CoV) is a large family of viruses that cause afflictions ranging from the common cold to more severe pathologies such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV). A novel coronavirus (nCoV) is a new strain that has now been identified in humans. The recognition of a new coronavirus identified in December 2019, named CoVID-19 are common for coronavirus researchers. Detailed investigations found that SARS Coronavirus-2 was initially transmitted from civets to humans and MERS-CoV from dromedary camels to humans. Advances in biology have resulted in a greater understanding of coronavirus, including them to adapt to new environments, trans-species infection and the emergence of new subtypes. New tools of cell and molecular biology have led to an increased understanding of intracellular replication and viral cell biology. Along with the advent of reverse genetic approaches in the past five years; it is now possible to begin to define the determinants of viral replication, trans-species adaptation, and human disease. The most progress has been made on SARS-CoV 2, highlighting specific structural requirements for its functions in the CoV life cycle as well as mechanisms behind its pathogenesis. In this review, we will provide a through insight to the life cycle of CoV, its genetics, replication process and reverse genetic applications to SCoV along with advances in its research. This review aims to establish the current knowledge on CoV-2 by highlighting the recent progress that has been made and comparing it to previous knowledge. We also conclude with a brief discussion on practices to decrease risk factors for transmission and treatment options.

Species identification of Trichinella originated from various host and different geographical location by MALDI-TOF

The foodborne zoonotic nematode Trichinella spp. can cause human trichinellosis when raw or undercooked contaminated meat is ingested.To date, twelve Trichinella species/genotypes have been described. According to EU regulation any Trichinella larvae detected during mandatory routine examinations need to be identified at a species level by a competent laboratory. Currently, Trichinella species identification is performed using molecular biology tools such as multiplex PCR, PCR-sequencing or PCR-RFLP. These techniques require high level of skills for good interpretation of the results. Due to its rapidness and ease of use a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protocol was previously developed for the identification of Trichinella species.Using this method, spectra from different Trichinella species and strains were acquired allowing to generate new Main Spectra (MSP). Finally a new MSP database from Trichinella spp. Samples of different countries (France, Germany and Poland), including field samples, was generated. Comparing the different main spectra, Trichinella worms were identified at the species level and differences in the genetic diversities within the different species are discussed.In conclusion, using the previously described method on field samples is a reliable, rapid, easy-to-use and cheap tool for Trichinella species identification. The new Trichinella database could be incremented with new samples. It constitutes a tool, which could be used as an alternative method to replace the actual molecular methods for Trichinella species identification.

Investigating the specific role of external load on the performance versus stability trade-off in microbial fuel cells

The performance and behavior of microbial fuel cells (MFCs) are influenced by among others the external load (Rext). In this study, the anode-surface biofilm formation in MFCs operated under different Rext selection/tracking-strategies was assessed. MFCs were characterized by electrochemical (voltage/current generation, polarization tests, EIS), molecular biological (microbial consortium analysis) and bioinformatics (principal component analysis) tools. The results indicated that the MFC with dynamic Rext adjustment (as a function of the actual MFC internal resistance) achieved notably higher performance but relatively lower operational stability, mainly due to the acidification of the biofilm. The opposite (lower performance, increased stability) could be observed with the static (low or high) Rext application (or OCV) strategies, where adaptive microbial processes were assumed. These possible adaptation phenomena were outlined by a theoretical framework and the significant impact of Rext on the anode colonization process and energy recovery with MFCs was concluded.

Keratin 19 Promotes Cell Cycle by Regulating GSK3B‐Cyclin D3 Pathway in Breast Cancer Cells

Keratin 19 (K19) is a cytoskeletal protein that is utilized as a diagnostic and prognostic marker in select human carcinomas. Despite the positive correlation between higher expression of K19 in tumor and worse patient survival, the role of K19 in breast cancer remains unclear. We previously used K19 knockout (KO) MCF7 breast cancer cells to identify that K19 is required for proper cell cycle progression. Specifically, K19 regulated expression of D‐type cyclins (cyclin D1 and cyclin D3) which promote the G1/S transition. We found that K19 interacts with cyclin D3, and a loss of K19 resulted in decreased protein stability of cyclin D3. To better understand how K19 prevents cyclin D3 degradation, we examined the cyclic adenosine 3′,5′‐monophosphate (cAMP) signaling pathway where cAMP induction causes glycogen synthase kinase GSK3B to promote cyclin D3 degradation. When cells were treated with forskolin to elevate intracellular cAMP level, cyclin D3 turnover was increased in the absence of K19, suggesting that K19 inhibits GSK3B from degrading cyclin D3. Indeed, inhibiting GSK3B activity using LiCl and GSK3B expression via siRNA rescued the cyclin D3 level in K19 KO cells. These data suggest that K19 plays an important role in GSK3B‐mediated cyclin D3 degradation. By using molecular biology tools and biochemical approaches, we are in the process of characterizing specifics of K19‐dependent regulation of cyclin D3 stability including identification of GSK3B and K19 domains responsible for their interaction. We aim to provide evidences for a novel mechanism by which a cytoskeletal protein stabilizes a cell cycle regulator and promotes proper cell cycle progression.Schematic model for the role of K19 in regulation of GSK3B‐mediated cyclin D3 degradation.(a) K19 serve as scaffold for both for GSK3B and cyclin D3, preventing GSK3B to access cyclin D3.(b) In absence of K19, GSK3B has a full access to cyclin D3 for degradation.Figure 1

Jurkat T‐cells chronically growing at acidic pH 7.2 show increased CO2 hydration activity

In our previous study (Vega et al. Faseb J. 32, S787.14) we reported that a clone of Jurkat T‐cell line that we have been chronically propagating at pH 7.2 (10% CO2) showed a significantly different growth pattern than the control clone at pH 7.4 (5% CO2).Since carbonic anhydrase (CA) is a key contributor to cellular pH regulation, here we tested if pH7.2 and pH7.4 clones have a different CA activity that was measured with the Wilbur‐Anderson assay; briefly, one unit is defined as 2*(T0‐T)/T, where T0 and T are the times in seconds in the absence and presence of enzyme, respectively, required for a saturated CO2 solution (4 mL) to lower pH of 20 mM TRIS buffer (6 mL) from 8.3 to 6.3 at 0°C.In a control experiment, CA with the reported activity (≥ 3,000 units/mg dry weight) showed in duplicate experiments 3,048 (T0=50.5 sec, T=10.5 sec, 0.0025 mg, determined with Hanna HI10832) and 3,111 (T0=66 sec, T=13.5 sec, 0.0025 mg, determined with a timer).To avoid the osmotic shock to the cells, we added 0.85% NaCl to all the assay’s solutions and tested if the presence of 0.85% NaCl affects CA activity.In the presence of 0.85% NaCl of NaCl, the values were 1,869±95 (T0=72.3±0.9 sec, T=21.8±0.8 sec, n=5), both blank T0 and enzyme T times being slower most likely because CO2 content in the saturated aqueous solution was smaller due to the presence of another solute, 0.85% NaCl.For the experiment, cell suspensions were span twice in the TRIS (20 mM)+NaCl 0.85% buffer at 0°C to achieve 1 mL of 10 million cells/mL density that was added to 5 mL of the TRIS buffer (pH 8.3), then immediately 4 mL of 0.85% NaCl saturated with CO2 was added, and time for pH to drop from 8.3 to 6.3 was measured; the final cell count being 10 million/mL in 10 mL of the reaction volume.For the pH7.4 clone, T= 82.3±1.1 sec (n=6) was slower than blank determination in 0.85% NaCl resulting in the apparent negative CA activity of −0.24 formally calculated as 2*(T0‐T)/T; while for the pH7.2 clone, the T= 77.3±1.4 sec (n=6) resulted in −0.13 (p&lt;0.05).If the increased CO2 hydration in the pH7.2 clone is caused by the increase in CA molecules alone, we would expect that these cells would have an additional 0.11/(10*106 cells)/(3000 units/mg)/(1000 mg/g)/(30,000 g/mole) * 6.022*1023 = 7.4*104 CA molecules per cell.We conclude that 1) isotonic NaCl significantly inhibits apparent CA activity in the context of Wilbur‐Anderson assay units, 2) both pH7.2 and 7.4 clones at 1 million/mL cell density inhibited CO2 hydration compared to the blank CO2 hydration in the Wilbur‐Anderson assay supplemented with 0.85% NaCl; 3) in relation to each other, pH7.2 clone has a higher CO2 hydration activity than pH7.4 clone speculatively due to estimated additional 7.4*104 CA molecules.Differential CA activity and expression in the pH7.2 and 7.4 clones will be further compared using physiology and molecular biology tools.