Whole genome sequencing of simmental cattle for SNP and CNV discovery.

BACKGROUD: The single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) are two major genomic variants, which play crucial roles in evolutionary and phenotypic diversity. RESULTS: In this study, we performed a comprehensive analysis to explore the genetic variations (SNPs and CNVs) of high sperm motility (HSM) and poor sperm motility (PSM) Simmental bulls using the high-coverage (25×) short-read next generation sequencing and single-molecule long reads sequencing data. A total of ~ 15 million SNPs and 2,944 CNV regions (CNVRs) were detected in Simmental bulls, and a set of positive selected genes (PSGs) and CNVRs were found to be overlapped with quantitative trait loci (QTLs) involving immunity, muscle development, reproduction, etc. In addition, we detected two new variants in LEPR, which may be related to the artificial breeding to improve important economic traits. Moreover, a set of genes and pathways functionally related to male fertility were identified. Remarkably, a CNV on SPAG16 (chr2:101,427,468 - 101,429,883) was completely deleted in all poor sperm motility (PSM) bulls and half of the bulls in high sperm motility (HSM), which may play a crucial role in the bull-fertility. CONCLUSIONS: In conclusion, this study provides a valuable genetic variation resource for the cattle breeding and selection programs.

Two-Color CRISPR Imaging Reveals Dynamics of Herpes Simplex Virus 1 Replication Compartments and Virus-Host Interactions.

Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring an ~600-bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. CRISPR-assisted single viral genome tracking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split green fluorescent protein (GFP) in amplifying fluorescent signals, dSaCas9-tdTomato10x and dSpCas9-GFP14x were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently come into contact with each other but do not mix, suggesting that RC territory is highly stable. Last, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR-Cas9-based imaging tools to study DNA viruses in living cells. IMPORTANCE Herpes simplex virus 1 (HSV-1), a representative of the family Herpesviridae, is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin. A common viral replication strategy is to form RCs that concentrate factors required for viral replication. Efficient strategies for imaging the dynamics of viral genomes, RC formation, and the interaction between the virus and host offer the opportunity to dissect the steps of the infection process and determine the mechanism underlying each step. We have developed an efficient two-color imaging system based on CRISPR-Cas9 technology to detect HSV-1 genomes quantitatively in living cells. Our results shed light on novel aspects of RC dynamics and virus-host interactions.

Aerosolizable Plasmid DNA Dry Powders Engineered by Thin-film Freezing.

PURPOSE: This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery. METHODS: Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) with various drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e., mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF)) were determined, and selected powders were used for further characterization. RESULTS: Of the nine dry powders prepared, their MMAD values were about 1-2 µm, with FPF values (delivered) of 40-80%. The aerosol properties of the powders were inversely correlated with the pDNA loading and the solid content in the pDNA solution before TFF. Powders prepared with Tris-EDTA buffer or cosolvents (i.e., 1,4-dioxane or tert-butanol in water), instead of water, showed slightly reduced aerosol properties. Ultimately, powders prepared with pDNA loading at 5% (w/w), 0.25% of solid content, with or without Tris-EDTA were selected for further characterization due to their overall good aerosol performance. The pDNA powders exhibited a porous matrix structure, with a moisture content of < 2% (w/w). Agarose gel electrophoresis confirmed the chemical integrity of the pDNA after it was subjected to TFF and after the TFF powder was actuated. A cell transfection study confirmed that the activity of the pDNA did not change after it was subjected to TFF. CONCLUSION: It is feasible to use TFF to produce aerosolizable pDNA dry powder for pulmonary delivery, while preserving the integrity and activity of the pDNA.

Juglone promotes antitumor activity against prostate cancer via suppressing glycolysis and oxidative phosphorylation.

The treatments currently used for prostate cancer (PC) do not meet clinical needs, and thus, new therapies with greater effectiveness are urgently required. Metabolic reprogramming of tumor cells is emerging as an exciting field for cancer therapy. Although the Warburg effect is a common feature of glucose metabolism in many cancers, PC cells have a unique metabolic phenotype. Non-neoplastic prostate cells show reduced oxidative phosphorylation (OXPHOS) because large, accumulated zinc inhibits citrate oxidation. During transformation, there are low levels of zinc in PC cells, and the tricarboxylic acid (TCA) cycle is reactivated. However, metastatic PC exhibits the Warburg effect. Due to metabolic differences in prostate tissue, targeting metabolic alterations in PC cells is an attractive therapeutic strategy. In this study, we investigated the effect of juglone on energy metabolism in PC cells. We found that juglone inhibited cell proliferation and induced apoptosis. Mechanistically, we demonstrated that juglone suppressed OXPHOS and glycolysis due to its inhibition of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) activity. Furthermore, downregulation of PFK and PK, but not HK contributed to the inhibition of these enzyme activities. The current study indicates that further development of juglone for PC treatment would be beneficial.

Genomic analysis reveals a KIT-related chromosomal translocation associated with the white coat phenotype in yak.

White coat pigmentation is a striking phenotype of many domesticated species and has various genetic controls. The Tianzhu White yak, an indigenous breed with a complete white coat, has fascinated Tibetans for centuries. However, the genetic basis of this trait remains unknown. Here, we conducted population genomics analysis and genome-wide association study based on the whole-genome sequencing data of 38 white and 59 non-white-coated yak. The results revealed the presence of KIT-linked Cs alleles characterized by the translocations between chromosomes 6 and 29 in all-white yak. Furthermore, structural variations showed additional duplications of the Cs alleles in white yak compared with colour-sidedness cattle. Interestingly, the Cs alleles associated with the white coat phenotype in yak were found to have introgressed from taurine cattle. Our findings unveil the shared genetic control of the white coat phenotype and its evolution in closely related bovine species.

Viability of Lactobacillus acidophilus in Thin-Film Freeze-Dried Powders Filled in Delayed-Release Vegetarian Capsules in a Simulated Gastric Fluid.

Previously, we have shown that thin-film freeze-drying can be applied to prepare dry powders of bacteria such as Lactobacillus acidophilus. Herein, we tested the viability of L. acidophilus in thin-film freeze-dried powders (TFF powders) filled in delayed-release vegetarian capsules in a simulated gastric fluid (SGF) consisting of 0.1N hydrochloric acid and sodium chloride. Initially, we determined the water removal rate from frozen thin films on relatively larger scales (i.e., 10-750 g). We then prepared and characterized two TFF powders of L. acidophilus with either sucrose and maltodextrin or sucrose and hydroxypropyl methylcellulose acetate succinate (HPMC-AS), a pH-sensitive polymer, as excipients and evaluated the viability of the bacteria after the TFF powders were filled in delayed-release vegetarian capsules and the capsules were incubated in the SGF for 30 min. On 10-750 g scales and at the settings specified, water removal from frozen thin films was faster than from slow shelf-frozen bulk solids. When the L. acidophilus in sucrose and HPMC-AS TFF powder was filled into a delayed-release capsule that was placed into another delayed-release capsule, the bacterial viability reduction after incubation in the SGF can be minimized to within 1 log in colony forming unit (CFU). However, for the L. acidophilus in sucrose and maltodextrin TFF powder, even in the capsule-in-capsule dosage form, bacterial CFU reduction was > 2 logs. TFF powders of live microorganisms containing an acid-resistant material in capsule-in-capsule delayed-release vegetarian capsules have the potential for oral delivery of those microorganisms.

TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells.

A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.

Factors Limiting the Translatability of Rodent Model-Based Intranasal Vaccine Research to Humans.

The intranasal route of vaccination presents an attractive alternative to parenteral routes and offers numerous advantages, such as the induction of both mucosal and systemic immunity, needle-free delivery, and increased patient compliance. Despite demonstrating promising results in preclinical studies, however, few intranasal vaccine candidates progress beyond early clinical trials. This discrepancy likely stems in part from the limited predictive value of rodent models, which are used frequently in intranasal vaccine research. In this review, we explored the factors that limit the translatability of rodent-based intranasal vaccine research to humans, focusing on the differences in anatomy, immunology, and disease pathology between rodents and humans. We also discussed approaches that minimize these differences and examined alternative animal models that would produce more clinically relevant research.

Immunogenicity of Antigen Adjuvanted with AS04 and Its Deposition in the Upper Respiratory Tract after Intranasal Administration.

Adjuvant system 04 (AS04) is in injectable human vaccines. AS04 contains two known adjuvants, 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and insoluble aluminum salts. Data from previous studies showed that both MPL and insoluble aluminum salts have nasal mucosal vaccine adjuvant activity. The present study was designed to test the feasibility of using AS04 as an adjuvant to help nasally administered antigens to induce specific mucosal and systemic immunity as well as to evaluate the deposition of antigens in the upper respiratory tract when adjuvanted with AS04. Alhydrogel, an aluminum (oxy)hydroxide suspension, was mixed with MPL to form AS04, which was then mixed with ovalbumin (OVA) or 3× M2e-HA2, a synthetic influenza virus hemagglutinin fusion protein, as an antigen to prepare OVA/AS04 and 3× M2e-HA2/AS04 vaccines, respectively. In mice, AS04 enabled antigens, when given intranasally, to induce specific IgA response in nasal and lung mucosal secretions as well as specific IgG response in the serum samples of the immunized mice, whereas subcutaneous injection of the same vaccine induced specific antibody responses only in the serum samples but not in the mucosal secretions. Splenocytes isolated from mice intranasally immunized with the OVA/AS04 also proliferated and released cytokines (i.e., IL-4 and IFN-γ) after in vitro stimulation with the antigen. In the immunogenicity test, intranasal OVA/AS04 was not more effective than intranasal OVA/MPL at the dosing regimens tested. However, when compared to OVA/MPL, OVA/AS04 showed a different atomized droplet size distribution and more importantly a more favorable OVA deposition profile when atomized into a nasal cast that was 3-D printed based on the computer tomography scan of the nose of a child. It is concluded that AS04 has mucosal adjuvant activity when given intranasally. In addition, there is a reason to be optimistic about using AS04 as an adjuvant to target an antigen of interest to the right region of the nasal cavity in humans for immune response induction.

Intranasal delivery of a nicotine vaccine candidate induces antibodies in mouse blood and lung mucosal secretions that specifically neutralize nicotine.

OBJECTIVE: Cigarette smoking is one of the leading causes of death in the world. The majority of the smokers have tried to quit, but only a few of them were able to achieve long-term abstinence, due to the high addictiveness of nicotine. Nicotine-specific antibodies have the potential to block the euphoric effect of nicotine by forming antibody-antigen complexes in the blood circulation. Since nicotine is taken largely by inhalation, inducing anti-nicotine antibodies in lung and nasal mucosal secretions, in addition to blood circulation, is expected to be beneficial. SIGNIFICANCE: The importance of this study is to establish the feasibility of inducing nicotine-neutralizing antibodies not only in the blood, but also in the lung and nasal mucosal secretions, by intranasal administration of a nicotine vaccine candidate. METHODS: Nicotine-keyhole limpet hemocyanin conjugate (Nic-KLH) was prepared and mixed with monophosphoryl lipid A (MPL) as an adjuvant. Nic-KLH/MPL was given intranasally or subcutaneously to mice, and the titers, affinity, and specificity of the nicotine-specific antibodies in nasal and lung mucosal secretions and blood samples were determined using (competitive) ELISA. RESULTS: Nasal Nic-KLH/MPL immunization elicited robust nicotine-specific neutralizing IgA in mouse nasal and lung secretions, in additional to anti-nicotine IgG in blood circulation. The nicotine-specific IgG level in mice nasally immunized with Nic-KLH/MPL was lower than in mice subcutaneously immunized with the same Nic-KLH/MPL, but a heterologous prime-boost immunization strategy helped to increase it. CONCLUSION: Intranasal immunization with a nicotine vaccine candidate can induce systemic and mucosal antibodies that specifically neutralize nicotine.

The COVID-19 Vaccine Race: Challenges and Opportunities in Vaccine Formulation.

In the race for a safe and effective vaccine against coronavirus disease (COVID)-19, pharmaceutical formulation science plays a critical role throughout the development, manufacturing, distribution, and vaccination phases. The proper choice of the type of vaccine, carrier or vector, adjuvant, excipients, dosage form, and route of administration can directly impact not only the immune responses induced and the resultant efficacy against COVID-19, but also the logistics of manufacturing, storing and distributing the vaccine, and mass vaccination. In this review, we described the COVID-19 vaccines that are currently tested in clinical trials and provided in-depth insight into the various types of vaccines, their compositions, advantages, and potential limitations. We also addressed how challenges in vaccine distribution and administration may be alleviated by applying vaccine-stabilization strategies and the use of specific mucosal immune response-inducing, non-invasive routes of administration, which must be considered early in the development process.

ATL3 gene mutation in a Chinese family with hereditary sensory neuropathy type 1F.

Hereditary sensory neuropathy (HSN) comprises a group of progressive peripheral neuropathies predominantly affecting the sensory nerves. To date, two different ATL3 gene mutations have been reported to be responsible for HSN type 1F (HSN1F). Here, we report a family in which the members presented numbness of the lower limbs and recurrent foot ulceration. Symptoms of foot ulcers disappeared in the years after onset, which suggests that the family members showed benign and mild symptoms compared with the affected patients reported previously. Laboratory examinations and electrophysiological data suggested axonal degeneration of the peripheral sensory nerves, while motor neurons were not involved. Exome sequencing revealed the previously reported c.C1013G (p.Pro338Arg) mutation of the ATL3 gene. This is the first report of ATL3 mutation in Chinese patients with HSN. Cells expressing mutant ATL3 exhibited disruption of the endoplasmic reticulum network, suggesting a dominant-negative effect. There was no significant difference in the expression of the endoplasmic reticulum stress marker binding immunoglobulin protein (BiP) between cells expressing wild-type or mutant ATL3. Further studies are required to ascertain the relevance of the changes in endoplasmic reticulum morphology to axonal degeneration of sensory nerves.

Giant urethral calculus in anterior urethral diverticulum: a case report.

BACKGROUND: In this case report, giant calculus in the urethral diverticulum was found through ureteroscopy investigation, the pneumatic lithotripsy combined with ultrasound lithotripsy (PLCUL) was successfully performed to break down this rare and giant urethral calculus in the diverticulum without open surgery. CASE PRESENTATION: A 82-year-old male presented to the urology department, complaining of frequent urination and dysuria. One giant, dark brown stone (6.5 × 6 × 5.5 cm) was revealed in the diverticulum of the anterior urethra using combination of local ultrasound, pelvic Computer Tomography (CT) and Magnetic Resonance Imaging (MRI). The stone was then successfully broken down via the PLCUL, and the emptied anterior urethral diverticulum was left untreated. In the 18 months' follow-up, no new calculus was found in urethral tract, anterior diverticula became gradually smaller, eventually disappeared. CONCLUSION: In the treatment of giant calculus in the urethral diverticulum, this case report provides an effective method of lithotripsy in the clinical trials.

Development of novel 3D printable inks for protein delivery.

The application of 3D printing technology in the delivery of macromolecules, such as proteins and enzymes, is limited by the lack of suitable inks. In this study, we report the development of novel inks for 3D printing of constructs containing proteins while maintaining the activity of the proteins during and after printing. Different ink formulations containing Pluronic F-127 (20-35 %, w/v), trehalose (2-10 %, w/v) or mannitol, poly (ethylene glycol) diacrylate (PEGDA) (0 or 10 %, w/w), and diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO, 0 or 0.2 mg/mL) were prepared for 3D-microextrusion printing. The F2 formulation that contained ß-galactosidase (ß-gal) as a model enzyme, Pluronic F-127 (30 %), and trehalose (10 %) demonstrated the desired viscosity, printability, and dose flexibility. The shear-thinning property of the F2 formulation enabled the printing of ß-gal containing constructs with a good peak force during extrusion. After 3D printing, the enzymatic activity of the ß-gal in the constructs was maintained for an extended period, depending on the construct design and storage conditions. For instance, there was a 50 % reduction in ß-gal activity in the two-layer constructs, but only a 20 % reduction in the four-layer construct (i.e., 54.5 ± 1.2 % and 82.7 ± 9.9 %, respectively), after 4 days of storage. The ß-gal activity in constructs printed from the F2 formulation was maintained for up to 20 days when stored in sealed bags at room temperatures (21 ± 2 °C), but not when stored unsealed in the same conditions (e.g., ∼60 % activity loss within 7 days). The ß-gal from constructs printed from F2 started to release within 5 min and reached 100 % after 20 min. With the design flexibility offered by the 3D printing, the ß-gal release from the constructs was delayed to 3 h by printing a backing layer of ß-gal-free F5 ink on the constructs printed from the F2 ink. Finally, ovalbumin as an alternative protein was also incorporated in similar ink compositions. Ovalbumin exhibited a release profile like that of the ß-gal, and the release can also be modified with different shape design and/or ink composition. In conclusion, ink formulations that possess desirable properties for 3D printing of protein-containing constructs while maintaining the protein activity during and after printing were developed.

Effect of lipid composition on RNA-Lipid nanoparticle properties and their sensitivity to thin-film freezing and drying.

A library of 16 lipid nanoparticle (LNP) formulations with orthogonally varying lipid molar ratios was designed and synthesized, using polyadenylic acid [poly(A)] as a model for mRNA, to explore the effect of lipid composition in LNPs on (i) the initial size of the resultant LNPs and encapsulation efficiency of RNA and (ii) the sensitivity of the LNPs to various conditions including cold storage, freezing (slow vs. rapid) and thawing, and drying. Least Absolute Shrinkage and Selection Operator (LASSO) regression was employed to identify the optimal lipid molar ratios and interactions that favorably affect the physical properties of the LNPs and enhance their stability in various stress conditions. LNPs exhibited distinct responses under each stress condition, highlighting the effect of lipid molar ratios and lipid interactions on the LNP physical properties and stability. It was then demonstrated that it is feasible to use thin-film freeze-drying to convert poly(A)-LNPs from liquid dispersions to dry powders while maintaining the integrity of the LNPs. Importantly, the residual moisture content in LNP dry powders significantly affected the LNP integrity.Residual moisture content of ≤ 0.5% or > 3-3.5% w/w negatively affected the LNP size and/or RNA encapsulation efficiency, depending on the LNP composition. Finally, it was shown that the thin-film freeze-dried LNP powders have desirable aerosol properties for potential pulmonary delivery. It was concluded that Design of Experiments can be applied to identify mRNA-LNP formulations with the desired physical properties and stability profiles. Additionally, optimizing the residual moisture content in mRNA-LNP dry powders during (thin-film) freeze-drying is crucial to maintain the physical properties of the LNPs.

Epidermal growth factor: Expression in goat endometrial epithelia during early pregnancy and regulation by interferon tau and FOXO1.

In ruminants, establishment and maintenance of pregnancy depends upon a well-coordinated interaction between the conceptus and the maternal endometrium. Epidermal growth factor (EGF) is important for embryo implantation and pregnancy establishment. However, the regulatory mechanisms of EGF expression remain unclear. FOXO1, a member of the Forkhead box O (FOXO) subfamily of transcription factors, is currently accepted as a novel endometrial receptivity marker for humans and mice owing to its timely and specific expression at the window of implantation. In this study, we examined the spatiotemporal expression profile of EGF in goat uterus during early pregnancy (Day 0 to Day 50 of pregnancy) and verified that EGF expression was regulated by FOXO1 and interferon tau (IFNT). Our results showed that EGF was highly expressed in the luminal epithelium (LE) and the glandular epithelium (GE) during conceptus adhesion (Day 16 to Day 25 of pregnancy). After implantation, EGF protein signals were continuously detected in the endometrial epithelia and appeared in the conceptus trophectoderm. Furthermore, EGF expression could be up-regulated by IFNT in goat uterus and primary endometrial epithelium cells (EECs). The luciferase assay results showed that FOXO1 could promote EGF transcription by binding to its promoter. And FOXO1 positively regulates EGF expression in goat EECs. These findings contribute to better understanding the role and regulation mechanisms of EGF during ruminant early pregnancy.

Intranasal delivery of thin-film freeze-dried monoclonal antibodies using a powder nasal spray system.

Monoclonal antibodies (mAbs) administered intranasally as dry powders can be potentially applied for the treatment or pre-exposure prevention of viral infections in the upper respiratory tract. However, a method to transform the mAbs from liquid to dry powders suitable for intranasal administration and a device that can spray the dry powders to the desired region of the nasal cavity are needed to fully realize the potentials of the mAbs. Herein, we report that thin-film freeze-dried mAb powders can be sprayed into the posterior nasal cavity using Aptar Pharma's Unidose (UDS) Powder Nasal Spray System. AUG-3387, a human-derived mAb that neutralizes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was used in the present study. First, we prepared thin-film freeze-dried AUG-3387 powders (i.e., TFF AUG-3387 powders) from liquid formulations containing different levels of mAbs. The TFF AUG-3387 powder with the highest solid content (i.e., TFF AUG-3387C) was then chosen for further characterization, including the evaluation of the plume geometry, spray pattern, and particle size distribution after the powder was sprayed using the UDS Powder Nasal Spray. Finally, the deposition patterns of the TFF AUG-3387C powder sprayed using the UDS Powder delivery system were studied using 3D-printed nasal replica casts based on the CT scans of an adult and a child. It is concluded that it is feasible to intranasally deliver mAbs as dry powders by transforming the mAbs into dry powders using thin-film freeze-drying and then spraying the powder using a powder nasal spray system.

Inhalable dry powders of a monoclonal antibody against SARS-CoV-2 virus made by thin-film freeze-drying.

Monoclonal antibodies (mAbs) represent a promising modality for the prevention and treatment of viral infections. For infections that initiate from the respiratory tract, direct administration of specific neutralizing mAbs into lungs has advantages over systemic injection of the same mAbs. Herein, using AUG-3387, a human-derived mAb with high affinity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its various variants, as a model mAb, we formulated the mAb into dry powders by thin-film freeze-drying, confirmed that the AUG-3387 mAb reconstituted from the dry powders retained their integrity, high affinity to the SARS-CoV-2 spike protein receptor binding domain (RBD), as well as ability to neutralize RBD-expressing pseudoviruses. Finally, we showed that one of the AUG-3387 mAb dry powders have desirable aerosol properties for pulmonary delivery into the lung. We concluded that thin-film freeze-drying represents a viable method to prepare inhalable powders of virus-neutralizing mAbs for pulmonary delivery into the lung.

Accelerated water removal from frozen thin films containing bacteria.

Freeze-drying, or lyophilization, is widely used to produce pharmaceutical solids that contain temperature-sensitive materials. Herein, using Escherichia coli as a model live organism, whose viability in dry powders is highly sensitive to the water content in the powders, we demonstrated that the drying rate from the frozen thin films generated by thin-film freezing (TFF) is significantly faster than from the bulk frozen solids in conventional shelf freeze-drying. This is likely because the loosely stacked frozen thin films provided a larger solid-air interface and the low thickness of the thin films provided a low mass transfer resistance. The highly porous microstructure and high specific surface area of the thin-film freeze-dried powders may also be related to the faster drying observed. Moreover, we demonstrated that TFF can be applied to produce dry powders of E. coli, a Gram-negative bacterium, or Lactobacillus acidophilus, a Gram-positive bacterium, with minimum bacterial viability loss (i.e., within one log reduction). It is concluded that the TFF technology is promising in accelerating water removal from frozen samples.