Cold Chain Storage Research Articles

Trientine Tetrahydrochloride, From Bench to Bedside: A Narrative Review.

Trientine tetrahydrochloride (TETA-4HCl, Cuvrior®) is a copper chelating agent with the active moiety triethylenetetramine (trientine), developed by Orphalan, Inc. to address the unmet needs in the treatment of Wilson disease. The journey from bench to bedside builds upon the documented safety profile of trientine hydrochloride capsules developed initially to meet the needs of individuals intolerant to D-penicillamine (DPA). Trientine hydrochloride capsules are inherently unstable requiring strict cold chain storage conditions from production, transportation, and use at home by the patient. Trientine tetrahydrochloride has a distinctive, patent-protected unique polymorphic form, which permits the production at scale of film-coated scored tablets deemed room temperature stable for 36 months. Trientine tetrahydrochloride is supported by a well-characterized pharmacodynamic, pharmacokinetic, and metabolic profile demonstrating reliable and predictable dose linearity and dose proportionality kinetics. Trientine tetrahydrochloride is the only trientine formulation that has been compared with DPA in a prospective randomized clinical trial, demonstrating non-inferiority to DPA in adults with stable Wilson disease. On 28 April, 2022, the US Food and Drug Administration approved TETA-4HCl for use in adult patients with Wilson disease who are de-coppered and tolerant to DPA. Health authorities in multiple countries worldwide have approved TETA-4HCl for the treatment of adults and children aged 5 years or more who are intolerant to DPA including the European Union, UK, Saudi Arabia, Switzerland, Colombia, Australia, New Zealand, and China. This article aims to provide a comprehensive narrative review of the key milestones in the development of TETA-4HCl.

Optimization Strategies of Phase Change Materials Applied in Fruit Cold Chain: Apples Transportation of Freshippo

The current fruit cold chain in China faces problems such as low coverage of cold chain services, high turnover losses, and waste of resources. It has been found that novel temperature control technologies using phase change materials (PCM) can be applied to fruit cold chains but have not yet been widely adopted. This study attempts to predict the effectiveness of PCM in the two main stages of cold chain storage and transport based on real case scenarios. By assessing the economic, environmental, and social benefits achieved, this study examines whether PCM has practical value in optimizing the fruit cold chain for sustainable development. In this more detailed version, the study delves into the application of PCM technology in the fruit cold chain and its economic, environmental and social impacts. It presents additional case studies and theoretical discussions to provide more in-depth thinking and guidance for the sustainable development of the cold chain industry.

B-224 Rapid, Extraction-Free, and Portable Molecular Detection of HIV-1 and HCV Directly From Whole Blood

Abstract Background Early diagnosis of human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is key to preventing viral transmission and improving linkage to treatment. Isothermal amplification methods, such as reverse transcription looped-mediated amplification (RT-LAMP), have enabled molecular diagnostics to expand beyond centralized laboratories. Despite the advances in molecular point-of-care (POC) testing, many of these tests are costly and still depend on a reliable power-source, specialized equipment, and cold-chain storage, making them impractical for resource-limited settings. To overcome these barriers, there is a need for innovative molecular diagnostics for blood-borne pathogens. Here, we propose a simple, extraction-free, portable workflow for detecting HIV-1 and HCV from whole blood within 40 minutes. Methods We developed singleplex RT-LAMP-based tests using modified published primer sets. Contrived whole blood samples containing HIV-1 or HCV virions were diluted in equal parts water and loaded directly into optimized RT-LAMP master mixes. To mitigate cold-chain storage dependence, RT-LAMP reactions were performed using a lyophilized master mix. The reactions were heated for 30 minutes using a hand-held, battery-powered heating device for simultaneous virion lysis and amplification. For simple visual detection, amplification was coupled with a lateral flow-based dipstick test. The analytical sensitivity of each detection test was evaluated using contrived whole blood samples ranging in HIV-1 and HCV 1a viral loads. Additionally, HCV detection was evaluated on genotypes 1b, 2b, and 3a. For result confirmation, a custom CRISPR-Cas12a-based assay was used to verify the presence or absence of pathogen-specific amplicons following amplification. Results The diagnostic workflow can be completed within 40 minutes, including less than 10 minutes of hands-on time. At optimal conditions, the HIV-1 and HCV 1a singleplex tests had lower limits of detections of 4.89 log10 cp/mL and 3.77 log10 IU/mL, respectively. Evaluation of the published literature showed that these viral loads are within the ranges observed during acute HIV-1 and HCV infection. HCV detection was also observed for genotypes 1b and 3a, albeit at a lower sensitivity than genotype 1a. No detection was observed for genotype 2b, presumably due to the lower viral loads of the samples. No cross-reactivity between the two tests was observed. Conclusions Our proposed workflow allows for rapid detection of blood-borne pathogens without the need for complex equipment or cold-chain storage, showing potential for application in resource-limited settings. Additional validation within a clinical setting using fingerstick blood remains to be tested.

Development of cashew-alginate microbeads and powdered dose forms: prospects for oral vaccine delivery in chickens

ABSTRACT Conventional oral vaccine delivery in poultry is challenging due to vaccine degradation in the gastrointestinal (GI) environment and the need for cold-chain storage. Microencapsulation offers a solution by protecting vaccines from GI degradation and improving stability. Natural polymers like alginate and cashew gum have mucoadhesive properties, making them promising candidates for oral vaccine delivery. This study developed cashew-alginate microbeads and a powdered dose form for oral vaccine delivery in chickens. The microbeads were created using ionotropic gelation, while the powdered form was obtained via freeze-drying. These formulations were characterized for size, shape, and stability using scanning electron microscopy (SEM), light microscopy, X-ray diffraction (XRD), and Energy Dispersive X-ray (EDX). Peak adhesion time (PAT) was determined using chicken intestinal and esophageal tissues, and antigenicity was assessed with in-vitro hemagglutination (HA) and hemagglutination inhibition (HI) assays. The microbeads exhibited a spherical shape with a porous structure, suggesting enhanced antigen accommodation. Hemagglutination Inhibition tests indicated that the experimental vaccine remained effective without cold-chain storage for three months. These findings suggest that cashew-alginate microbeads are promising for oral vaccine delivery in poultry.

Equivalence of freeze-dried and liquid-frozen formulations of MVA-BN as smallpox and mpox vaccine

ABSTRACT Modified Vaccinia Ankara Bavarian Nordic (MVA-BN) as a smallpox and mpox vaccine has been approved in its liquid-frozen (LF) formulation in the US, Canada, and EU. A freeze-dried (FD) formulation may offer additional benefits, such as a longer shelf life and reduced dependence on cold chain storage and transport. In a phase 2 clinical trial, 651 vaccinia-naïve participants were vaccinated with two doses of MVA-BN LF or FD, 4 weeks apart. The objectives were to compare MVA-BN FD with LF in terms of vaccine-induced immune responses, safety, and reactogenicity. Non-inferiority of the immune response was assessed by the 95% CI of the geometric mean ratios. Both formulations induced robust vaccinia-specific humoral and cellular immune responses. At peak humoral responses (Week 6), geometric means of total antibody titers were 1096 (95% CI 1013, 1186) from the FD group and 877 (95% CI 804, 956) from the LF group, achieving the primary endpoint of non-inferiority of MVA-BN FD compared to MVA-BN LF. At peak cellular responses (Week 2), geometric means of T cell spot forming units were 449 (95% CI 341, 590) from the FD group and 316 (95% CI 234, 427) from the LF group. Both formulations of MVA-BN were well tolerated, with similar unsolicited AEs and solicited systemic reactions in both groups but slightly more local reactions in the FD group. No vaccine-related serious adverse events (SAEs) or vaccine-related AE of special interest were reported. The FD formulation of MVA-BN was shown to be equivalent to MVA-BN LF.

Quantitative PCR assays as a monitoring tool for bacterial genera in fresh fish fillets

Fresh fish fillets are a valuable but highly perishable food, and their rapid microbial deterioration is a drawback for food safety and sustainability of aquaculture, food and retail industries. Quantitative PCR (qPCR) assays based on 16S rRNA gene (16S) sequences were developed for the most abundant bacteria genera detected by metagenomics in fresh or processed fish fillets. The efficiency and specificity of six qPCR assays (for 16S of all bacteria or genera Shewanella, Pseudomonas, Carnobacterium, Janthinobacterium and Massilia) was verified using in silico predictions, cloning, sequencing and phylogenetic analyses of amplicons obtained from refrigerated control or high-pressure processed (HPP) European sea bass (Dicentrarchus labrax) fillets. In HPP sea bass fillets, significant decreases in total bacteria 16S and of Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium 16S compared to control fillets were confirmed by qPCR, after 11 days of refrigerated storage. The qPCR assays were successfully applied to monitor microbial contamination during refrigerated storage of fresh fillets from commercial (retail) sea bass and gilthead sea bream (Sparus aurata). Significant increases in total bacterial and Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium contamination were detected after 7–14 days. 16S copy number for total bacteria and the four target genera positively correlated with total viable counts using culture enumeration. 16S of Massilia, that is abundant in fresh fish fillets, did not significantly change during storage. The six validated qPCR assays developed are proposed as specific, sensitive, culture-independent methods for monitoring quality or processing outcomes for fish fillets during cold chain storage.

Low-cost multispectral sensor reveals cold chain breaks, meat type, and storage time in chicken meat samples

A portable and low-cost spectrometric system has been proposed to discriminate cold chain (frozen) interruption during the supply chain, as well as, the sample type among four different tested chicken meat and products (nugget, wing, thigh and drumstick) and the storage time elapsed after the cold chain break. To achieve this, an AS7265x sensor chipset was used and controlled by a custom programmed microcontroller through a Qwiic I2C interface. Communication between the user and the sensor setup was made using a custom programmed interface developed using the Python. Frozen chicken samples were thawed under various temperature-time combinations (4, 8, 16, and 24 h at 4 and 24 °C) and then refrozen at -18 °C. The refrozen samples were stored for up to 60 days. Spectral measurements (410–940 nm) were taken before thawing and at 1, 10, 30 and 60 days of storage after refreezing. The spectra obtained were analysed using Principal Component Analysis and outliers were eliminated using Mahalobobis distance. Several classification models (including Decision Tree, Random Forest, AdaBoost, Support Vector Machine, k-Nearest Neighbors, Gaussian-Naïve Bayes, Multilayer Perceptron, Gaussian Process, Linear and Quadratic Discriminant Analysis and Partial Least Squares Discriminant Analysis) were trained where appropriate. The top models with the highest predictive ability were selected and merged to build Soft Voting Classifiers (SVC) for each feature to be predicted. The SVC models correctly classified the samples with 93%, 92% and 83% accuracy for cold chain interruption, chicken meat/product type and storage time after thaw-refreeze cycle, respectively. In addition, a graphical interface was also developed so that it can be used by end users for food safety concerns. The results showed the potential of the developed low-cost sensor to rapidly detect the potential food safety risks due to cold chain breakage and to trace back the problem by predicting the storage time after refreezing.

Ratiometric Readout of Bacterial Infections via a Lyophilized CRISPR-Cas12a Sensor with Color-Changeable Bioluminescence.

The healthcare burden imposed by bacterial infections demands robust and accessible diagnostic methods that can be performed outside hospitals and centralized laboratories. Here, we report Pathogen Assay with Ratiometric Luminescence (PEARL), a sensitive and easy-to-operate platform for detecting pathogenic bacteria. The PEARL leveraged a color-changeable CRISPR-Cas12a sensor and recombinase polymerase amplification to elicit ratiometric bioluminescence responses to target inputs. This platform enabled robust and visualized identification of attomolar bacteria genome deoxyribonucleic acid according to the color changes of the reactions. In addition, the components of the color-changeable Cas12a sensor could be lyophilized for 3 month storage at ambient temperature and then be fully activated with the amplicons derived from crude bacterial lysates, reducing the requirements for cold-chain storage and tedious handling steps. We demonstrated that the PEARL assay is applicable for identifying the infections caused by Pseudomonas aeruginosa in different clinical specimens, including sputa, urines, and swabs derived from wounds. These results revealed the potential of PEARL to be used by untrained personnel, which will facilitate decentralized pathogen diagnosis in community- and resource-limited regions.

Exploring the Use of Ambientally Stored Methylene Diphosphonate Radiopharmaceutical Aliquots in Solving Challenging Situations in Developing Countries

Abstract Objectives The primary aim was to evaluate the prolonged quality characteristics of methyl diphosphonate (MDP) aliquots during ambient storage over a specified duration. This study further investigated potential additives that could enhance the stability of MDP aliquots stored under such conditions. Materials and Methods This was a laboratory-based experimental study conducted at the University Teaching Adult Hospital in Lusaka, Zambia. A total of 36 MDP aliquots stored at ambient conditions and 4 MDP aliquots stored at conventional refrigerated frozen conditions were labeled with technitium-99m (99mTc) and tested for radiochemical purity (RCP) and other quality characteristics. A comparative analysis of the stability and quality of MDP aliquots from the two cohorts was then conducted. Statistical Analysis Stata 14 was used to analyze the data on the RCP of all MDP aliquots. Results The RCP of ambient stored MDP aliquots was found to be ranging from 98 to 99%, while that for frozen and refrigerated ones ranged from 99 to 100%. There was also a 1% increase in RCP for both cohorts with argon gas purging (98 and 99%, respectively). Conclusion The RCP of MDP aliquots from both cohorts was much higher than the required minimum of 90% implying that there was no significant association of their stability and quality with the mode of storage. However, purging with argon gas seemed to increase the stability further in both streams. The study findings show potential for application in resource-constrained environments and centers, especially in developing countries, where challenges to maintain the cold storage chain of these important radiopharmaceuticals are likely to be encountered due to power outages.

I LAGH (ormoni della crescita long-acting): cosa sono e a chi proporli?

Children with growth hormone deficiency are currently treated with daily injections of recombinant growth hormone. The burden due to 365 injections per year and cold chain storage causes a poor medication adherence. Long-acting GH was developed to decrease injection frequency and to therefore promote a better therapeutic compliance. In this paper, we discuss about which long-acting drugs are currently on the market, their mechanism of action and which groups of patients may benefit from them.

Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry-state protein storage.

Protein biotherapeutics typically require expensive cold-chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold-chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., "cryoprotection" and "lyoprotection"), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake-forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1-16) were extracted via ethanol precipitation. PG size was generally consistent at ~70-100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1-16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, β-galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate-buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein-PG13 mixtures had a cake-like appearance and all frozen protein-PG13 mixtures had a Tg' of ~ -26°C. The lyoprotection and cake-forming properties of PG13 were density-dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

Changes in meat quality, metabolites and microorganisms of mutton during cold chain storage

During the cold chain storage process, changes in metabolites and microorganisms are highly likely to lead to changes in meat quality. To elucidate the changes in the composition of metabolites and microbiota during cold chain storage of mutton, this study utilized untargeted metabolome and 5R 16S rRNA sequencing analyses to investigate the changes in the longissimus dorsi under different cold chain temperatures (4 °C and −20 °C). With the extension of cold chain storage time, the meat color darkened and the content of C18:2n-6, C20:3n-6, and C23:0 were significantly increased in mutton. In this study, nine metabolites, including 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, alanylphenylala-nine, indole-3-acrylic acid and the others, were significantly altered during cold chain storage. The abundance of the dominant microorganisms, including Brachymonas, Aeromonas, Corynebacterium and Steroidobacter, was significantly altered. Furthermore, a high correlation was observed between the different metabolites and microorganisms. These findings provide an in-depth understanding of the effects of different cold chain storage temperatures and times on the quality of mutton.

System design and clinical validation of intelligent nucleic acid POCT for un SARS-CoV-2 detection within 30 min

Rapid and accurate point-of-care-test (POCT) allows us to substantially accelerate taking clinical decisions and implement strategic planning at the national level of preventative measures in epidemic prevention and control. Here, we report Intelligent Nucleic Acid Test (iNAT) for the RT-qPCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with a limit of detection (LOD) of 22.4 copies/mL. iNAT is a sample-to-answer system consisting of an instrument and a fully enclosed detection chip containing reaction reagents, based on rotary valve liquid control technology, ultrasonic-assisted nucleic acid extraction (NAE) technology, thin-sheet rapid PCR technology, freeze-drying technology, liquid transfer, NAE and RT-qPCR are all completed automatically, quickly and efficiently, and free of cold chain transportation and storage. The user only needs to put the chip with 160 μL sample into the instrument and click to start, the test result will report after 30 min. In a clinical trial between the iNAT and the traditional RT-qPCR kits approved by National Medical Products Administration (NMPA), involving 158 throat swab samples, the concordance rate reached 96.2%, the positive percent agreement (PPA) is 95.8%, and the negative percent agreement (NPA) is 97.3%, the parallel analysis of Ct values of 115 positive samples was a high correlation (R2 = 0.95), 4 of 10 weak positive samples were missed by iNAT while 6 of 10 weak positive samples were missed by reference kits. In a word, iNAT system provide an ultra-high sensitivity and fast POCT detection method for SARS-CoV-2.

Dry and liquid formulations of IBT-V02, a novel multi-component toxoid vaccine, are effective against Staphylococcus aureus isolates from low-to-middle income countries.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs.

Broad antibody and T cell responses to Ebola, Sudan, and Bundibugyo ebolaviruses using mono- and multi-valent adjuvanted glycoprotein vaccines

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion (“IVAX-1”) was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.

Hydrogen Bond-Mediated Self-Shielded Moisture-Responsive Structural Color for Time-Temperature Indicating.

Effective monitoring of the time-temperature history of biological reagents, chemical drugs, and perishable foods during cold chain storage is crucial for ensuring their quality and efficacy. Time-temperature indicators (TTIs) are developed to assess the cumulative impact of time and temperature on product quality. However, current indicators face challenges related to complex wrapping procedures, narrow tracking ranges, susceptibility to photobleaching, and pre-use instability, hampering widespread use. Herein, the first moisture-responsive 1D photonic crystal (1DPC) TTIs featuring robust structural colors, customizable time-temperature ranges, and reliable renewability are demonstrated. The indicators exhibit distinct color-changing responsiveness toward water vapor, which remains observable after prolonged storage at low temperatures. Significantly, the moisture responsiveness gradually diminishes at elevated temperatures over time due to ambient water-induced hydrogen bond formation, effectively shielding the indicator from external stimuli. This property enables the naked-eye inspection of product efficacy during cold chain storage. Additionally, the endowed flexibility of the TTI facilitates its easy attachment to targets, functioning as a convenient indicator label. Remarkably, the indicator can be stably stored for an extended period at room temperature before use, thereby showcasing substantial market potential.

In situ-formed cryomicroneedles for intradermal cell delivery

Cryomicroneedles (cryoMNs) offer a convenient and minimally invasive way to precisely deliver therapeutic cells intradermally for treating local and systemic diseases. cryoMNs are manufactured by shaping and freezing the cell-containing cryogenic media in a microneedle template, which allows cells to be packaged in advance for direct usage in the clinic. However, the current cryoMNs require cold-chain transportation and storage and do not permit the loading of autologous cells in situ. This article introduces the second generation of cryoMNs (S-cryoMNs) that address these limitations. Specifically, S-cryoMNs are made by dipping a porous MN scaffold in the cell suspension before cryopreservation. The porous scaffold can be transported at room temperature, and researchers can load any cells with the optimized cryogenic medium. As a proof-of-concept, we examined the loading and intradermal delivery of three cell types in clinically relevant in vitro and in vivo models, including mesenchymal stem cells for wound healing, melanocytes for vitiligo treatment, and antigen-pulsed dendritic cells for cancer vaccination.

Stability of Genotube® Swabs for African Swine Fever Virus Detection Using Loop-Mediated Isothermal (LAMP) Laboratory Testing on Samples Stored without Refrigeration.

African swine fever (ASF) is a transboundary viral disease which causes high mortality in pigs. In many low- and middle-income countries and in remote areas where diagnostic surveillance for ASF virus (ASFV) is undertaken, access to trained animal health technicians, sample collection, cold chain storage and transport of samples to suitably equipped laboratories can be limiting when traditional sampling and laboratory tests are used. Previously published studies have demonstrated that alternative sampling matrices such as swabs and filter papers can be tested using PCR without refrigeration for up to a week. This study used Genotube® swabs stored in temperate and tropical climates without refrigeration for four weeks after collection to demonstrate there was no change in test performance and results using loop-mediated isothermal amplification (LAMP) ASFV detection on a series of pig serum samples including serum spiked with a synthetic ASFV positive control, naturally acquired ASFV positive serum from Timor-Leste and negative ASFV serum samples. The use of Genotube® swabs for ASFV detection for surveillance purposes, coupled with testing platforms such as LAMP, can provide an alternative to traditional testing methodology where resources are limited and time from collection to testing of samples is prolonged.

Water-modulated low-wavelength fluorescence emission of PSmOrange

The fluorescent protein (FP) family has been a prominent focus for several decades due to its extensive applications in bioimaging. However, the fluorescence mechanisms of fluorescent proteins have not been fully elucidated. Recently, there have been reports suggesting the involvement of water in the fluorescent behaviors of FPs. Although numerous theoretical calculations and a few experimental studies based on protein solutions have been conducted, direct experimental evidence to unravel the role of water within fluorescent proteins remains elusive. Herein, we reported the impact of water on the low-wavelength fluorescence emission of PSmOrange, a model FP known for its excellent photostability and high-water content. We found that the presence of water induces the emergence of this low-wavelength fluorescence emission. This effect can be attributed to a hydrogen bond network involving the fluorophore and water molecules. Manipulating this hydrogen bond network by altering temperature or water types proved to be an effective strategy for modulating the fluorescent behaviors of PSmOrange. This study sheds light on the role of water in modulating the low-wavelength fluorescence emission of FPs and pave the way for the design and development of the next generation biomolecule-based fluorescence probes in real-time temperature monitoring in cold chain storage.

Optimized biomimetic minerals maintain activity of mRNA complexes after long term storage

mRNA therapeutics can be readily designed, manufactured, and brought to scale, as demonstrated by widespread global vaccination against COVID-19. However, mRNA therapies require cold chain shipment and storage from manufacturing to administration, which may limit them to affluent communities. This problem could be addressed by mimicking the known ability of mineralized fossils to durably stabilize nucleic acids under extreme conditions. We synthesized and screened 40 calcium-phosphate minerals for their ability to store and maintain the activity of lyophilized mRNA complexes. The optimal mineral formulation incorporated mRNA complexes with high efficiency (77 %), and increased mRNA transfection efficiency by 5.6-fold. Lyophilized mRNA complexes stored with the optimized mineral formulation for 6 months at 25 °C were 3.2-fold more active than those stored with state-of-the-art excipients, but without a mineral. mRNA complexes stored with minerals at room temperature did not decline in transfection efficacy from 3 days to 6 months of storage, indicating that minerals can durably maintain activity of therapeutic mRNA complexes without cold chain storage. Statement of significanceTherapeutic mRNA, such as mRNA COVID-19 vaccines, require extensive cold chain storage that limits their general application. This work screened a library of minerals to maintain the activity of mRNA complexes with freeze-drying. The optimized mineral was able to maintain mRNA activity up to 6 months of storage at room temperature outperforming current methods of freeze-drying therapeutic mRNA complexes.