A causal relationship between gut microbiota and subcortical brain structures contributes to the microbiota-gut-brain axis: a Mendelian randomization study.

A correlation between gut microbiota and brain structure, referring to as a component of the gut-brain axis, has been observed in observational studies. However, the causality of this relationship and its specific bacterial taxa remains uncertain. To reveal the causal effects of gut microbiota on subcortical brain volume, we applied Mendelian randomization (MR) studies in this study. Genome-wide association study data were obtained from the MiBioGen Consortium (n = 18,340) and the Enhancing Neuro Imaging Genetics through Meta-Analysis Consortium (n = 13,170). The primary estimate was obtained utilizing the inverse-variance weighted, while heterogeneity and pleiotropy were assessed using the Cochrane Q statistic, MR Pleiotropy RESidual Sum and Outlier, and MR-Egger intercept. Our findings provide strong evidence that a higher abundance of the genus Parasutterella is causally correlated with a decrease in intracranial volume (ß = -30,921.33, 95% CI -46,671.78 to -15,170.88, P = 1.19 × 10-4), and the genus FamilyXIIIUCG001 is associated with a decrease in thalamus volume (ß = -141.96, 95% CI: -214.81 to -69.12, P = 1.0× 10-4). This MR study offers novel perspectives on the intricate interplay between the gut microbiota and subcortical brain volume, thereby lending some support to the existence of the microbiota-gut-brain axis.

Proteomics Identifies LUC7L3 as a Prognostic Biomarker for Hepatocellular Carcinoma.

Alternative splicing has been shown to participate in tumor progression, including hepatocellular carcinoma. The poor prognosis of patients with HCC calls for molecular classification and biomarker identification to facilitate precision medicine. We performed ssGSEA analysis to quantify the pathway activity of RNA splicing in three HCC cohorts. Kaplan-Meier and Cox methods were used for survival analysis. GO and GSEA were performed to analyze pathway enrichment. We confirmed that RNA splicing is significantly correlated with prognosis, and identified an alternative splicing-associated protein LUC7L3 as a potential HCC prognostic biomarker. Further bioinformatics analysis revealed that high LUC7L3 expression indicated a more progressive HCC subtype and worse clinical features. Cell proliferation-related pathways were enriched in HCC patients with high LUC7L3 expression. Consistently, we proved that LUC7L3 knockdown could significantly inhibit cell proliferation and suppress the activation of associated signaling pathways in vitro. In this research, the relevance between RNA splicing and HCC patient prognosis was outlined. Our newly identified biomarker LUC7L3 could provide stratification for patient survival and recurrence risk, facilitating early medical intervention before recurrence or disease progression.

Interaction Behavior between Coarse and Fine Particles in the Reverse Flotation of Fluorapatite and Dolomite.

The precondition for efficient flotation of middle- to low-grade phosphate ore is the dissociation of apatite from gangue minerals through fine grinding. However, fine gangue minerals, such as dolomite, have a lower hardness and are thus easily overground and converted into slime during the grinding stage. The effect of -10 µm dolomite fines (Dol-10µm) on the flotation of -75 + 25 µm dolomite (Dol-75+25µm) and -75 + 25 µm fluorapatite (FAp-75+25µm) from a detailed mechanism was investigated by microflotation tests, cryo-electron microscopy analysis, contact angle measurements, bubble-particle attachment test, and EDLVO theory. It was found that Dol-10µm significantly reduced the recovery of Dol-75+25µm and FAp-75+25µm. The Dol-10µm particles could decrease the flotation recovery of Dol-75+25µm particles by masking on their surfaces, thus reducing the hydrophobicity of mineral surfaces and preventing them from floating. Furthermore, Dol-10µm particles were easily adsorbed on the FAp-75+25µm surface and could not be effectively floated, which results in fine dolomite being mixed into the concentrate at the bottom of the tank, thereby reducing the selectivity of reverse flotation for phosphate ore. The interaction energy calculation showed that the presence of NaOL led to an attractive interaction force between Dol-10µm and Dol-75+25µm, as well as between Dol-10µm and FAp-75+25µm. As a result, Dol-10µm particles presented a nonselective aggregation with Dol-75+25µm and FAp-75+25µm particles, explaining the phenomenon of "slime coating" and the depressing effect of dolomite fines on phosphate reverse flotation.

Two Quantum Proxy Blind Signature Schemes Based on Controlled Quantum Teleportation.

We present a scheme for teleporting an unknown, two-particle entangled state with a message from a sender (Alice) to a receiver (Bob) via a six-particle entangled channel. We also present another scheme for teleporting an unknown one-particle entangled state with a message transmitted in a two-way form between the same sender and receiver via a five-qubit cluster state. One-way hash functions, Bell-state measurements, and unitary operations are adopted in these two schemes. Our schemes use the physical characteristics of quantum mechanics to implement delegation, signature, and verification processes. Moreover, a quantum key distribution protocol and a one-time pad are adopted in these schemes.

Separability Criteria Based on the Weyl Operators.

Entanglement as a vital resource for information processing can be described by special properties of the quantum state. Using the well-known Weyl basis we propose a new Bloch decomposition of the quantum state and study its separability problem. This decomposition enables us to find an alternative characterization of the separability based on the correlation matrix. We show that the criterion is effective in detecting entanglement for the isotropic states, Bell-diagonal states and some PPT entangled states. We also use the Weyl operators to construct an detecting operator for quantum teleportation.

A unique B cell epitope-based particulate vaccine shows effective suppression of hepatitis B surface antigen in mice.

OBJECTIVE: This study aimed to develop a novel therapeutic vaccine based on a unique B cell epitope and investigate its therapeutic potential against chronic hepatitis B (CHB) in animal models. METHODS: A series of peptides and carrier proteins were evaluated in HBV-tolerant mice to obtain an optimised therapeutic molecule. The immunogenicity, therapeutic efficacy and mechanism of the candidate were investigated systematically. RESULTS: Among the HBsAg-aa119-125-containing peptides evaluated in this study, HBsAg-aa113-135 (SEQ13) exhibited the most striking therapeutic effects. A novel immunoenhanced virus-like particle carrier (CR-T3) derived from the roundleaf bat HBV core antigen (RBHBcAg) was created and used to display SEQ13, forming candidate molecule CR-T3-SEQ13. Multiple copies of SEQ13 displayed on the surface of this particulate antigen promote the induction of a potent anti-HBs antibody response in mice, rabbits and cynomolgus monkeys. Sera and purified polyclonal IgG from the immunised animals neutralised HBV infection in vitro and mediated efficient HBV/hepatitis B virus surface antigen (HBsAg) clearance in the mice. CR-T3-SEQ13-based vaccination induced long-term suppression of HBsAg and HBV DNA in HBV transgenic mice and eradicated the virus completely in hydrodynamic-based HBV carrier mice. The suppressive effects on HBsAg were strongly correlated with the anti-HBs level after vaccination, suggesting that the main mechanism of CR-T3-SEQ13 vaccination therapy was the induction of a SEQ13-specific antibody response that mediated HBV/HBsAg clearance. CONCLUSIONS: The novel particulate protein CR-T3-SEQ13 suppressed HBsAg effectively through induction of a humoural immune response in HBV-tolerant mice. This B cell epitope-based therapeutic vaccine may provide a novel immunotherapeutic agent against chronic HBV infection in humans.

Gadd45a is a heterochromatin relaxer that enhances iPS cell generation.

Reprogramming of somatic cells to induced pluripotent stem cells rewrites the code of cell fate at the chromatin level. Yet, little is known about this process physically. Here, we describe a fluorescence recovery after photobleaching method to assess the dynamics of heterochromatin/euchromatin and show significant heterochromatin loosening at the initial stage of reprogramming. We identify growth arrest and DNA damage-inducible protein a (Gadd45a) as a chromatin relaxer in mouse embryonic fibroblasts, which also enhances somatic cell reprogramming efficiency. We show that residue glycine 39 (G39) in Gadd45a is essential for interacting with core histones, opening chromatin and enhancing reprogramming. We further demonstrate that Gadd45a destabilizes histone-DNA interactions and facilitates the binding of Yamanaka factors to their targets for activation. Our study provides a method to screen factors that impact on chromatin structure in live cells, and identifies Gadd45a as a chromatin relaxer.

Structural and relative stabilities, electronic properties, and hardness of iron tetraborides from first prinicples.

First-principles calculations were carried out to investigate the structure, phase stability, electronic property, and roles of metallicity in the hardness for recently synthesized FeB4 with various different structures. Our calculation indicates that the orthorhombic phase with Pnnm symmetry is the most energetically stable one. The other four new dynamically stable phases belong to space groups monoclinic C2/m, orthorhombic Pmmn, trigonal R3̅m, and hexagonal P63/mmc. Their mechanical and thermodynamic stabilities are verified by calculating elastic constants, formation enthalpies, and phonon dispersions. We found that all phases are stabilized further under pressure. Above the pressure of about 50 GPa, the formation enthalpy of Pmmn is almost equal to that of P63/mmc phase. The analysis on density of states not only demonstrates that formation of strong covalent bonding in these compounds contributes greatly to their stabilities but also that they all exhibit metallic behavior which does not relate to the approach used. By considering metallic contributions, the estimated Vickers hardness values based on the semiempirical model show that the OsB4-structured FeB4, with a hardness of 48.1 GPa, well exceeding the limitation of superhardness (40 GPa), is more hard than the most stable phase. The others are predicted to be potential hard materials. Moreover, the atomic configuration and strong B-B covalent bonds are found to play important roles in the hardness of materials.

Phase stability, mechanical properties, hardness, and possible reactive routing of chromium triboride from first-principle investigations.

The first-principles calculations are employed to provide a fundamental understanding of the structural features and relative stability, mechanical and electronic properties, and possible reactive route for chromium triboride. The predicted new phase of CrB3 belongs to the rhombohedral phase with R-3m symmetry and it transforms into a hexagonal phase with P-6m2 symmetry at 64 GPa. The mechanical and thermodynamic stabilities of CrB3 are verified by the calculated elastic constants and formation enthalpies. Also, the full phonon dispersion calculations confirm the dynamic stability of predicted CrB3. Considering the role of metallic contributions, the calculated hardness values from our semiempirical method for rhombohedral and hexagonal phases are 23.8 GPa and 22.1 GPa, respectively. In addition, the large shear moduli, Young's moduli, low Poisson's ratios, and small B∕G ratios indicate that they are potential hard materials. Relative enthalpy calculations with respect to possible constituents are also investigated to assess the prospects for phase formation and an attempt at high-pressure synthesis is suggested to obtain chromium triboride.

Research Progress of Tamarixetin and its glycosides.

Tamarixetin and its glycosides are widely distributed in natural plants, and they are also natural flavonoid derivatives of quercetin. Its main pharmacological effects include antioxidant, anti-inflammatory, antiviral, anticancer, cardiovascular effects, etc. The pharmacokinetics showed that the distribution of direct absorption differed from that of biosynthesis. At the same time, research shows that tamarixetin is safe to use because it has little self-toxicity. In this paper, 181 articles on tamarixetin published from 1976 to 2023 are obtained from PubMed, China Knowledge Base Database, Wanfang Data, and other electronic databases. Tamarixetin is searched based on keywords, and 121 articles remain. Transformation synthesis, pharmacokinetics, pharmacological action, and structure-activity relationship of tamarixetin were reviewed.

WeChat-based remote follow-up management reduces the burden of home care and anxiety on parents of children with refractory epilepsy: A randomized controlled study.

OBJECTIVE: The present research was designed to study the effect of WeChat-based remote follow-up management on the burden of home care and anxiety on parents of children with refractory epilepsy. METHODS: 161 refractory epileptic children were included in this study. They were divided into control group and WeChat group according to their management protocols after discharge, namely, control group with traditional follow-up (n = 81) and WeChat group with remote follow-up based on WeChat (n = 81). We evaluated home care burden by family caregiver task Inventory (FCTI) scale and zarit burden interview (ZBI) scale, and evaluated negative emotion by self-rating anxiety Scale (SAS) scale and self-Rating depression scale (SDS) scale. RESULTS: There was no significant difference in the demographic characteristics of epileptic children and their parents and the scores of FCTI, ZBI, SAS and SDS before treatment between the 2 groups (all P > .05), and the score of FCTI (20.86 ± 4.26), ZBI (39.63 ± 4.46), SAS (44.49 ± 4.15) and SDS (50.02 ± 4.13) in WeChat group were all significantly lower than the score of FCTI (25.25 ± 3.71), ZBI (45.47 ± 4.61), SAS (52.75 ± 4.93) and SDS (54.51 ± 6.59) in control group (all P < .05). CONCLUSION: WeChat-based remote follow-up management reduces the burden of home care and anxiety on parents of children with refractory epilepsy.

Modulation of Skin Inflammatory Responses by Aluminum Adjuvant.

Aluminum salt (AS), one of the most commonly used vaccine adjuvants, has immuno-modulatory activity, but how the administration of AS alone may impact the activation of the skin immune system under inflammatory conditions has not been investigated. Here, we studied the therapeutic effect of AS injection on two distinct skin inflammatory mouse models: an imiquimod (IMQ)-induced psoriasis-like model and an MC903 (calcipotriol)-induced atopic dermatitis-like model. We found that injection of a high dose of AS not only suppressed the IMQ-mediated development of T-helper 1 (Th1) and T-helper 17 (Th17) immune responses but also inhibited the IMQ-mediated recruitment and/or activation of neutrophils and macrophages. In contrast, AS injection enhanced MC903-mediated development of the T-helper 2 (Th2) immune response and neutrophil recruitment. Using an in vitro approach, we found that AS treatment inhibited Th1 but promoted Th2 polarization of primary lymphocytes, and inhibited activation of peritoneal macrophages but not bone marrow derived neutrophils. Together, our results suggest that the injection of a high dose of AS may inhibit Th1 and Th17 immune response-driven skin inflammation but promote type 2 immune response-driven skin inflammation. These results may provide a better understanding of how vaccination with an aluminum adjuvant alters the skin immune response to external insults.

Establishing extended pluripotent stem cells from human urine cells.

BACKGROUND: Extended pluripotent stem cells (EPSCs) can contribute to both embryonic and trophectoderm-derived extraembryonic tissues. Therefore, EPSCs have great application significance for both research and industry. However, generating EPSCs from human somatic cells remains inefficient and cumbersome. RESULTS: In this study, we established a novel and robust EPSCs culture medium OCM175 with defined and optimized ingredients. Our OCM175 medium contains optimized concentration of L-selenium-methylcysteine as a source of selenium and ROCK inhibitors to maintain the single cell passaging ability of pluripotent stem cells. We also used Matrigel or the combination of laminin 511 and laminin 521(1:1) to bypass the requirement of feeder cells. With OCM175 medium, we successfully converted integration-free iPSCs from easily available human Urine-Derived Cells (hUC-iPSCs) into EPSCs (O-IPSCs). We showed that our O-IPSCs have the ability to form both intra- and extra- embryonic chimerism, and could contribute to the trophoblast ectoderm lineage and three germ layer cell lineages. CONCLUSIONS: In conclusion, our novel OCM175 culture medium has defined, optimized ingredients, which enables efficient generation of EPSCs in a feeder free manner. With the robust chimeric and differentiation potential, we believe that this system provides a solid basis to improve the application of EPSCs in regenerative medicine.

Serum neuronal pentraxin 2 is related to cognitive dysfunction and electroencephalogram slow wave/fast wave frequency ratio in epilepsy.

BACKGROUND: Cognitive dysfunction in epileptic patients is a high-incidence complication. Its mechanism is related to nervous system damage during seizures, but there is no effective diagnostic biomarker. Neuronal pentraxin 2 (NPTX2) is thought to play a vital role in neurotransmission and the maintenance of synaptic plasticity. This study explored how serum NPTX2 and electroencephalogram (EEG) slow wave/fast wave frequency ratio relate to cognitive dysfunction in patients with epilepsy. AIM: To determine if serum NPTX2 could serve as a potential biomarker for diagnosing cognitive impairment in epilepsy patients. METHODS: The participants of this study, conducted from January 2020 to December 2021, comprised 74 epilepsy patients with normal cognitive function (normal group), 37 epilepsy patients with cognitive dysfunction [epilepsy patients with cognitive dysfunction (ECD) group] and 30 healthy people (control group). The mini-mental state examination (MMSE) scale was used to evaluate cognitive function. We determined serum NPTX2 levels using an enzyme-linked immunosorbent kit and calculated the signal value of EEG regions according to the EEG recording. Pearson correlation coefficient was used to analyze the correlation between serum NPTX2 and the MMSE score. RESULTS: The serum NPTX2 level in the control group, normal group and ECD group were 240.00 ± 35.06 pg/mL, 235.80 ± 38.01 pg/mL and 193.80 ± 42.72 pg/mL, respectively. The MMSE score was lowest in the ECD group among the three, while no significant difference was observed between the control and normal groups. In epilepsy patients with cognitive dysfunction, NPTX2 level had a positive correlation with the MMSE score (r = 0.367, P = 0.0253) and a negative correlation with epilepsy duration (r = -0.443, P = 0.0061) and the EEG slow wave/fast wave frequency ratio value in the temporal region (r = -0.339, P = 0.039). CONCLUSION: Serum NPTX2 was found to be related to cognitive dysfunction and the EEG slow wave/fast wave frequency ratio in patients with epilepsy. It is thus a potential biomarker for the diagnosis of cognitive impairment in patients with epilepsy.

MiR-223-3p affects the proliferation and apoptosis of HCAECs in Kawasaki disease by regulating the expression of FOXP3.

OBJECTIVE: Kawasaki disease (KD) can lead to permanent damage to coronary structures, the pathogenesis of which remains unknown. This experiment was designed to investigate whether miR-223-3p secreted in the serum of KD patients affects the proliferation and apoptosis of HCAECs in KD by regulating FOXP3. METHODS: Blood samples were collected in acute febrile phase of KD, after IVIG treatment, and from healthy controls. Transfected into HCAECs cells by synthetic FOXP3 siRNA/NC. A co-culture system was established between HCAECs cells transfected with FOXP3 siRNA/NC and THP1 cells added with three sera. RESULTS: Compared with the control group, the expressions of miR-223-3p, RORγt, and Th17 in serum of KD patients were significantly upregulated, and the expressions of TGF-ß1, FOXP3 and Treg were significantly downregulated. At the same time, the levels of IL-6, IL-17, and IL-23 were significantly increased, and the levels of IL-10 and FOXP3 were significantly decreased. After IVIG treatment, the patient's above results were reversed. The serum of KD patients increased the expression of miR-223-3p and inhibited the expression of FOXP3 in HCAECs cells. IVIG serum is the opposite. Overexpression of miR-223-3p also promoted the apoptosis of HCAECs. In addition, serum from KD patients promoted apoptosis, whereas serum after IVIG treatment inhibited apoptosis. KD patient serum downregulated the expression of FOXP3, Bcl2, TGF-ß1 and IL-10 in cells, and upregulated the expression of caspase3, Bax, IL-17, IL-6, and IL-23. The opposite results were obtained with IVIG-treated sera. CONCLUSION: miR-223-3p secreted in serum of KD patients can regulate the expression of FOXP3 and affect the proliferation, apoptosis, and inflammation of cells.

[Surgical outcome and prognostic factors in focal cortical dysplasia with intractable epilepsy].

OBJECTIVE: To analyze surgical outcome and relevant surgical parameters including resection extent of epileptogenic zone,pathological subtype, brain MRS and MRI results in FCD with intractable epilepsy. METHODS: We retrospectively analyzed surgical outcomes of 35 patients with intractable epilepsy related to focal cortical dysplasia, accepted surgery in the first affiliated hospital of Fujian Medical University from January 2008 to January 2010, with 12-36 months of postoperative follow-up. The relevance between complete resection, pathological subtype, MRS and MRI result and surgical outcome were statistically evaluated. RESULTS: 22 patients (66.7%) were Engel class I, 5 patients (14.3%) were class II, 6 patients (17.2%) were class III, 2 patients (5.8%) were class IV. Complete resection of epileptogenic zone (P < 0.05), FCD type I (P < 0.05) correlated significantly with favorable surgical outcome. Other factors such as MRI results, abnormal NAA/CHO + Cr ratio on the contralateral side of epileptogenic zone, as well as MRS-accurate lateralization did not influence outcome. CONCLUSION: Overall, the surgical outcome of FCD is favorable. Complete resection, FCD type I correlates significantly with favorable surgical outcome.

Microtiter Plate-Based Differential Scanning Fluorimetry: A High-Throughput Method for Efficient Formulation Development.

Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ∼ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited.

Effect of Carbide Slag on Removal of Na+/K+ from Red Mud Based on Water Leaching.

Red mud (RM) is a hazardous solid waste discharged from the alumina production process. The stock of RM is very large, and it has strong alkalinity and certain radioactivity, which makes it have a very serious adverse effect on the environment. Many scholars have carried out extensive experimental investigations on the minimization, hazard-free treatment, and reutilization of RM, and encouraging results have been obtained. However, reutilization of RM has been restricted mainly due to its complex composition and strong alkalinity. In this study, carbide slag, a byproduct of acetylene production, was utilized to remove alkalis (Na+ and K+) from RM by calcium ion replacement. The effects of the temperature, liquid-to-solid ratio, carbide slag dose, and leaching time on dealkalization of RM by carbide slag were studied. The leaching mechanism of sodium was investigated and analyzed using inductively coupled plasma-atomic emission spectrometry, X-ray diffraction, and scanning electron microscopy with energy-dispersive spectrometry. Under the optimal conditions, the residual Na2O and K2O amount in the RM after dealkalization using the carbide slag diminished to less than 0.93 and 0.45 wt %. More than 78.80% of Na2O and 58.84% of K2O could be dissolved under the optimal conditions. The cancrinite structure in the initial RM was destroyed, and soluble sodium salts formed in the suspension can be easily replaced by carbide slag reducing Na+. The dealkalization process of RM by using carbide slag was controlled by chemical reaction of shrinking core model, where the apparent activation energy was 4.92 kJ/mol.

A prophylactic effect of aluminium-based adjuvants against respiratory viruses via priming local innate immunity.

Infection caused by respiratory viruses can lead to a severe respiratory disease and even death. Vaccination is the most effective way to prevent the disease, but it cannot be quickly applied when facing an emerging infectious disease. Here, we demonstrated that immunization with an aluminium-zinc hybrid particulate adjuvant (FH-001) alone, bearing great resemblance in morphology with commonly used aluminium-based adjuvants in vaccines, could quickly induce mice to generate a broadly protective immune response to resist the lethal challenge of influenza B viruses. Furthermore, a multi-omics-based analysis revealed that the alveolar macrophage and type I interferon pathway, rather than adaptive immunity and type II interferon pathway, were essential for the observed prophylactic effect of FH-001. More importantly, a similar protective effect was observed against influenza A virus strain A/Shanghai/02/2013(H7N9), A/California/04/2009(H1N1) and respiratory syncytial virus. Therefore, we introduced here a new and promising strategy that can be quickly applied during the outbreak of emerging respiratory viruses.

Capsid destabilization and epitope alterations of human papillomavirus 18 in the presence of thimerosal.

Thimerosal has been widely used as a preservative in drug and vaccine products for decades. Due to the strong propensity to modify thiols in proteins, conformational changes could occur due to covalent bond formation between ethylmercury (a degradant of thimerosal) and thiols. Such a conformational change could lead to partial or even complete loss of desirable protein function. This study aims to investigate the effects of thimerosal on the capsid stability and antigenicity of recombinant human papillomavirus (HPV) 18 virus-like particles (VLPs). Dramatic destabilization of the recombinant viral capsid upon thimerosal treatment was observed. Such a negative effect on the thermal stability of VLPs preserved with thimerosal was shown to be dependent on the thimerosal concentration. Two highly neutralizing antibodies, 13H12 and 3C3, were found to be the most sensitive to thimerosal treatment. The kinetics of antigenicity loss, when monitored with 13H12 or 3C3 as probes, yielded two distinctly different sets of kinetic parameters, while the data from both monoclonal antibodies (mAbs) followed a biphasic exponential decay model. The potential effect of thimerosal on protein function, particularly for thiol-containing proteinaceous active components, needs to be comprehensively characterized during formulation development when a preservative is necessary.