Efficient and cost-effective non-invasive population monitoring as a method to assess the genetic diversity of the last remaining population of Amur leopard (Panthera pardus orientalis) in the Russia Far East.

Small populations of the endangered species are more vulnerable to extinction and hence require periodic genetic monitoring to establish and revisit the conservation strategies. The Amur leopard is critically endangered with about 100 individuals in the wild. In this study, we developed a simple and cost-effective noninvasive genetic monitoring protocol for Amur leopards. Also, we investigated the impact of fecal sample's age, storage, and collection season on microsatellite genotyping success and data quality. We identified 89 leopard scats out of the 342 fecal samples collected from Land of the Leopard between 2014-2019. Microsatellite genotyping using 12 markers optimized in 3 multiplex PCR reactions reveals presence of at least 24 leopard individuals (18 males and 6 females). There was a significant difference in the success rate of genotyping depending on the time from feces deposition to collection (p = 0.014, Fisher's exact test), with better genotyping success for samples having <2 weeks of environmental exposure. Amur leopard genetic diversity was found low (Ho- 0.33, HE- 0.35, and NA- 2.57) with no visible population substructure and recent bottleneck signature. Although a historical bottleneck footprint was observed. Mitochondrial DNA diversity was also found low with two haplotypes differing by a point mutation reported in 1,769 bp of investigated sequence covering parts of cytochrome b gene (846 bp), NADH-5 gene (611 bp) and control region (312 bp). We recommend periodic genetic monitoring of wild Amur leopards following the proposed methodology to achieve cost effectiveness and efficiency.

Correction: Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits.

[This corrects the article DOI: 10.1371/journal.pone.0256869.].

Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits.

Human serum albumin (HSA) has been widely used as a pharmaceutical excipient in Botulinum toxin serotype A (BoNT/A) products that are indicated for use in therapeutics and cosmetics. However, HSA as a human-derived material has some concerns, such as the potential risk of transmission of infectious agents, an insufficient supply, and difficulty in maintaining a certain quality. For those reasons, newly developed BoNT/A products (CORETOX®, Medytox, Inc., Republic of Korea) contained polysorbate 20, a non-human-derived excipient, to replace the HSA. However, most safety studies of polysorbate 20 have been conducted with non-invasive routes of administration, and thus there are a few studies on the safety of polysorbate 20 when administered intramuscularly. To secure the in vivo safety profile of polysorbate 20, a four-week repeated intramuscular dose toxicity study (0.02, 0.1, and 0.4 mg/kg, one injection every two weeks for a total of three injections) was conducted in 66 Sprague-Dawley (SD) rats. An intradermal irritation study was further conducted with 18 New Zealand White (NZW) rabbits. The toxicological evaluation of HSA (0.06 and 0.12 mg/kg) was also carried out as a comparative substance. Systemic and local toxicities were not observed in any of the SD rats or NZW rabbits based on clinical signs, body weight, hematology, clinical biochemistry, macroscopic findings on necropsy, histopathology of the injection site, and allergic reactions. The current study suggested that intramuscular administration of polysorbate 20 was considered to be safe at a level similar to that of HSA, which has an in vivo safety profile accumulated over the years. This provided the basis for the in vivo safety profile of polysorbate 20 administered intramuscularly and the scientific reliability of the use of polysorbate 20 as an alternative to HSA, which is used as an excipient for various pharmaceuticals in terms of its safety.

Injection funnel persistence time and oolemma resistance during intracytoplasmic sperm injection and subsequent embryo development.

OBJECTIVE: This study aimed to investigate whether the injection funnel persistence time and oolemma resistance during the intracytoplasmic sperm injection (ICSI) are associated with subsequent embryo quality. DESIGN: A prospective observational study at a university hospital. METHODS: One hundred and twenty normal-appearing metaphase II oocytes were collected from 54 ICSI cycles. Injection funnel was observed at 0, 30, 60, and 90 s after ICSI, and the injection funnel persistence time was assigned to "no funnel," "0-30," "30-60," "60-90," and ">90 s." The degree of oolemma resistance during ICSI was recorded as "no," "mild," "moderate," and "severe." Subsequent embryos on day 3 after ICSI were evaluated morphologically, and formation of top-quality embryo and embryo score was assessed. We newly developed "oolemma score," based on the injection funnel persistence time and oolemma resistance, and the predictability of top-quality embryo was assessed. RESULTS: Among the five groups by injection funnel persistence time, the proportion of top-quality embryo and embryo score (64.3%, 32) was highest in the "30-60 s," but not significant. Among the four groups by oolemma resistance, the proportion of top-quality embryo and embryo score (53.7%, 32) was highest in "no group." The proportion of top-quality embryo in "no group" was significantly higher than "moderate group" (p = 0.012) and "severe group" (p = 0.043). The median embryo score in "no group" was significantly higher than "severe group" (p = 0.041). Newly developed "oolemma score" could predict well the formation of top-quality embryo with a statistical significance (cutoff >14.5, area under the curve 0.695, p < 0.001). CONCLUSION: Embryo quality or score is more closely associated with oolemma resistance during ICSI. New "oolemma score" would help to identify embryo developmental potential of each mature oocyte in ICSI cycles.

Cryomilling-induced solid dispersion of poor glass forming/poorly water-soluble mefenamic acid with polyvinylpyrrolidone K12.

The effect of mechanical impact on the polymorphic transformation of mefenamic acid (MFA) and the formation of a solid dispersion of mefenamic acid, a poor glass forming/poorly-water soluble compound, with polyvinylpyrrolidone (PVP) K12 was investigated. The implication of solid dispersion formation on solubility enhancement of MFA, prepared by cryomilling, was investigated. Solid state characterization was conducted using powder X-ray diffraction (PXRD) and Fourier-transform infrared (FTIR) spectroscopy combined with crystal structure analysis. Apparent solubility of the mixtures in pH 7.4 buffer was measured. A calculation to compare the powder patterns and FTIR spectra of solid dispersions with the corresponding physical mixtures was conducted. Solid state characterization showed that (1) MFA I transformed to MFA II when pure MFA I was cryogenically milled (CM); and (2) MFA forms a solid dispersion when MFA was cryogenically milled with PVP K12. FTIR spectral analysis showed that hydrogen bonding facilitated by mechanical impact played a major role in forming solid dispersions. The apparent solubility of MFA was significantly improved by making a solid dispersion with PVP K12 via cryomilling. This study highlights the importance of cryomilling with a good hydrogen bond forming excipient as a technique to prepare solid dispersion, especially when a compound shows a poor glass forming ability and therefore, is not easy to form amorphous forms by conventional method.

Organic vapor sorption method of isostructural solvates and polymorph of tenofovir disoproxil fumarate.

Tenofovir disoproxil fumarate (TDF) is a prodrug of tenofovir that belongs to a class of antiretroviral drugs, a nucleotide reverse transcriptase inhibitor. An acetonitrile solvate of TDF I, another new solvated form of TDF, was prepared and solid state characterization of its form was conducted using powder X-ray diffraction, FT-IR spectroscopy, and organic vapor sorption isotherm. During the characterization work, it was discovered that (1) TDF I can form solvates and polymorph with a wide variety of organic solvents as well as water and (2) to different extents, these solvates undergoes anisotropic lattice contraction/expansion during desolvation/solvation process suggesting the formation of isostructural solvates of TDF. Solvents used in this study include ethanol, isopropyl alcohol, acetonitrile, cyclohexane, toluene, and water. Four new solvates using ethanol, isopropyl alcohol, acetonitrile, and toluene vapor and one polymorph using water vapor were discovered. Their solid state characterizations were conducted using powder X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, and Fourier transform infrared spectroscopy. A variety of isostructural solvates and a polymorph of TDF was produced by an organic vapor sorption method, showing varying physicochemical properties. This study demonstrates an alternative crystallization method to obtain isostructural solvates.

Requirement of the coiled-coil domain of PML-RARalpha oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation.

Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-alpha (RARalpha) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(DeltaC-C)-RARalpha, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the DeltaC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the DeltaC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the DeltaC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARalpha. However, the RA-mediated induction of C/EBPbeta in cells expressing the DeltaC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation all contribute to the oncogenic activity of PML-RARalpha.

Ability of the human cytomegalovirus IE1 protein to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells.

In one of the earliest events in human cytomegalovirus (HCMV)-infected cells, the major immediate-early (IE) protein IE1 initially targets to and then disrupts the nuclear structures known as PML oncogenic domains (PODs) or nuclear domain 10. Recent studies have suggested that modification of PML by SUMO is essential to form PODs and that IE1 both binds to PML and may disrupt PODs by preventing or removing SUMO adducts on PML. In this study, we showed that in contrast to herpes simplex virus type 1 (HSV-1) IE110 (ICP0), the loss of sumoylated forms of PML by cotransfected IE1 was resistant to the proteasome inhibitor MG132 and that IE1 did not reduce the level of unmodified PML. Reduced sumoylation of PML was also observed in U373 cells after infection with wild-type HCMV and proved to require IE1 protein expression. Mutational analysis revealed that the central hydrophobic domain of IE1, including Leu174, is required for both PML binding and loss of PML sumoylation and confirmed that all IE1 mutants tested that were deficient in these functions also failed both to target to PODs and to disrupt PODs. These same mutants were also inactive in several reporter gene transactivation assays and in inhibition of PML-mediated repression. Importantly, a viral DNA genome containing an IE1 gene with a deletion [IE1(Delta290-320)] that was defective in these activities was not infectious when transfected into permissive fibroblast cells, but the mutant IE1(K450R), which is defective in IE1 sumoylation, remained infectious. Our mutational analysis strengthens the idea that interference by IE1 with both the sumoylation of PML and its repressor activity requires a physical interaction with PML that also leads to disruption of PODs. These activities of IE1 also correlate with several unusual transcriptional transactivation functions of IE1 and may be requirements for efficient initiation of the lytic cycle in vivo.

PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate-early IE2 protein of human cytomegalovirus.

The protein inhibitor of activated STAT1 (PIAS1), known to be a small ubiquitin-like modifier (SUMO) E3 ligase, was found to interact with the human cytomegalovirus IE2 protein. We found that the sumoylation of IE2 was markedly enhanced by wild-type PIAS1 but not by a mutant containing a Cys to Ser substitution at position 351 (C351S) within the RING finger-like domain. In target reporter gene assays, wild-type PIAS1, but not the C351S mutant, enhanced the IE2-mediated transactivations of viral polymerase promoter and cellular cyclin E promoter and this augmentation required the intact sumoylation sites of IE2. Our results suggest that PIAS1 acts as a SUMO E3 ligase toward IE2 and that it may regulate the transactivation function of IE2. To our knowledge, IE2 is the first viral target found to be regulated by a SUMO E3 ligase.