Characterization of HIF-1α Knockout Primary Human Natural Killer Cells Including Populations in Allogeneic Glioblastoma.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Natural killer cells (NK) are major innate effector cells with broad cytotoxicity against tumors. Accordingly, NK cells are ideal candidates for cancer immunotherapy, including glioblastoma (GBM). Hypoxia is a common feature of solid tumors, and tumor cells and normal cells adapt to the tumor microenvironment by upregulating the transcription factor hypoxia-inducible factor (HIF)-1α, which can be detrimental to anti-tumor effector immune cell function, including that of NK cells. We knocked out HIF-1α in human primary NK cells using clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9). Then, cellular characterizations were conducted in normoxic and hypoxic conditions. Electroporating two HIF-1α-targeting guide RNA-Cas9 protein complexes inhibited HIF-1α expression in expanded NK cells. HIF-1α knockout human NK cells, including populations in hypoxic conditions, enhanced the growth inhibition of allogeneic GBM cells and induced apoptosis in GBM-cell-derived spheroids. RNA-sequencing revealed that the cytotoxicity of HIF-1α knockout NK cells could be related to increased perforin and TNF expression. The results demonstrated that HIF-1α knockout human NK cells, including populations, enhanced cytotoxicity in an environment mimicking the hypoxic conditions of GBM. CRISPR-Cas9-mediated HIF-1α knockout NK cells, including populations, could be a promising immunotherapeutic alternative in patients with GBM.

Design, Regioselective Synthesis, and Photophysical Properties of Perfluoronaphthalene-Based Donor-Acceptor-Donor Fluorescent Dyes.

A one-step route to a series of perfluoronaphthalene-based donor (D)-acceptor (A)-D fluorescent dyes with various electron-donating groups was developed. The perfluoronaphthalene moiety in the D-A-D dyes served as a good electron-accepting aromatic ring with excellent intramolecular charge transfer properties, as determined by density functional theory calculations and measurements of the fluorescence properties in solution, in poly(methyl methacrylate) (PMMA) films, and in crystal form. Notably, replacing the naphthalene ring with perfluoronaphthalene in the D-A-D dyes carrying the phenothiazine moiety not only stabilized the highest occupied molecular orbital and lowest unoccupied molecular orbital energy levels but also reduced the energy band gap to change the emission color from blue to yellow. Among the four synthesized perfluoronaphthalene D-A-D dyes, those bearing diphenylamino groups afforded the best fluorescence quantum yields in Et2O solution (0.60) and in PMMA film (0.65) because the propeller structure of the diphenylamino group that acts as a donor substituent effectively suppresses radiation-free deactivation. In contrast, in the crystalline state, the carbazoyl-bearing D-A-D dye provided the best fluorescence quantum yield (0.35) because the radiation-free inactivation was suppressed by π-πF stacking at the donor site, which was confirmed by single-crystal X-ray analysis.

Synthesis of Small Fluorescent Molecules and Evaluation of Photophysical Properties.

A series of aniline-based fluorophores were newly synthesized. To increase their fluorescence quantum yields, it was particularly important to substitute 3,3,3-trifluoroprop-1-enyl (TFPE) groups next to the amino group to benefit from an extended π-electron delocalization. Among these, 5-CN-2-TFPE-aniline was found to behave as an excellent fluorophore with a reasonable fluorescence quantum yield of 0.89 even in aqueous solution. l-Alanine peptide, a nonfluorescent analogue of 5-CN-2-TFPE-aniline, was synthesized and successfully employed as an enzyme probe to detect aminopeptidase N activity.

Virilism and Ectopic Expression of HSD17B5 in Mature Cystic Teratoma.

Mature cystic teratoma (MCT) is rarely involved in the overproduction of steroid hormones in contrast to sex cord stromal tumors. A 31-year-old woman visited our hospital with hirsutism, hoarseness, and hair loss from the scalp. Serum testosterone and free-testosterone levels were 7.3 ng/ml and 2.3 pg/ml, respectively, which were markedly in excess of the age adjusted female standard levels. Basal blood levels of steroid hormones and serum levels of 17-hydroxyprogesterone at 1 h after intravenous injection of adrenocorticotropic hormone demonstrated that 21-hydroxylase deficiency was not the underlying cause of her virilization. A subsequent chromosomal test with G-banding revealed a karyotype of 46XX. Magnetic resonance imaging revealed a mass in the left ovary, which was subsequently diagnosed as MCT. Detailed pathological analysis of the tumor indicated that it was comprised of skin components, sweat glands, with hair and fat texture, glandular epithelium and fibrous connective tissue, consistent with the characteristic composition of MCT. Immunohistochemical analysis demonstrated marked immunoreactivity of 17beta-hydroxysteroid dehydrogenase (HSD17B5), an enzyme that can convert androstenedione to testosterone. Following surgical removal of the tumor, testosterone and free testosterone levels were markedly decreased (0.3 ng/ml and 0.4 pg/ml, respectively) and other symptoms abated. In conclusion, this is the first report of an ovarian MCT associated with clinical virilization caused by the ectopic production of testosterone possibly because of an overexpression of intratumoral HSD17B5.

Population Genetic Structure of QoI-Resistant Pestalotiopsis longiseta Isolates Causing Tea Gray Blight.

Resistance to the quinone outside inhibitor (QoI) fungicides in the tea gray blight-causing fungus Pestalotiopsis longiseta is a serious problem in Japanese tea cultivation. We conducted a population genetic analysis of QoI-resistant P. longiseta isolates on the Makinohara Plateau, Shizuoka Prefecture, Japan's largest tea-growing area, to elucidate the disease's epidemiology and the spread of QoI resistance. Inter simple sequence repeat (ISSR) analysis of 1,083 isolates from 395 fields collected from 2009 to 2012 detected 42 ISSR types, designated as PL01 to PL42. A total of 18, seven, and 38 ISSR types were detected in highly resistant, moderately resistant, and sensitive isolates, respectively. No distinct phylogenetic relationship corresponding to QoI sensitivity or sampling location was observed. No annual changes in the population genetic structure of highly resistant isolates were observed during the study period. A different ISSR type was predominant among QoI-resistant isolates in each region. Analysis of molecular variance revealed significant genetic differentiation in populations of highly resistant isolates among regions (FCT = 0.213) and farmers (FCT = 0.071). Consequently, we speculate that QoI-resistant P. longiseta strains occurred in a number of clonal lineages and spread by both natural and artificial transmission, such as rain splash and plucking machines, throughout each region on the Makinohara Plateau.

A biphenyl type two-photon fluorescence probe for monitoring the mitochondrial membrane potential.

Here we describe the design and synthesis of a bifunctional two-photon fluorescence probe, N,N'-|dimethyl-4,4'-(biphenyl-2,1-ethenediyl)dipyridinium hexafluorophosphate (BP6). HeLa, Hek293, and Paramecium caudatum cells were stained with BP6. BP6 accumulated on the mitochondria of all three cell types when the mitochondrial membrane potential was high. As the mitochondrial membrane potential decreased following the addition of carbonyl cyanide m-chlorophenyl hydrazine, BP6 moved from the mitochondria to the nucleus in a reversible manner, depending on the mitochondrial membrane potential status. The maximum value of the two-photon absorption cross-section of BP6 is 250 GM (1 GM=1×10(-50) cm(4) s molecules(-1) photon(-1)). This value is 3 and 30 times larger, respectively, than that of the conventional mitochondria selective probes, rhodamine 123 and green fluorescence protein. These results suggest that BP6 should be useful for monitoring mitochondrial membrane potential by two-photon excitation.

Traumatic intracranial aneurysm in a distal posterior cerebral artery: A case report and literature review.

Background: Traumatic intracranial aneurysms (TICAs) are rare and known to rupture easily and have a high mortality rate. Case Description: An 87-year-old male patient with no neurological deficits presented to our hospital after head trauma. Computed tomography (CT) revealed a tentorial acute subdural hematoma (ASDH). The patient was managed conservatively and discharged home six days after hospitalization. Two days later, the patient returned with a severe headache. CT showed that the ASDH had enlarged and extended from the tentorium to the convexity. CT angiography and digital subtraction angiography revealed a pseudoaneurysm in a branch of the left posterior inferior temporal artery. The patient was diagnosed with an enlarged ASDH due to a ruptured TICA that arose from the P3 segment. We performed endovascular intervention with parent artery occlusion (PAO) using n-butyl-2-cyanoacrylate (NBCA). The parent artery was accessed through the left posterior communicating artery because left vertebral angiography revealed an aplastic left P1 segment. After navigating the microcatheter near the aneurysm, we injected 33% NBCA into the parent artery. The pseudoaneurysm disappeared after injection. The patient was discharged on hospital day 25 despite persistent delirium. Conclusion: This is the first report of a TICA arising from the P3 segment that was treated with PAO using NBCA. TICAs are rare; however, a TICA must be considered when an enlarged hematoma caused by head injury is detected.

Minimum ten-year results of a porous acetabular component for Crowe I to III hip dysplasia using an elevated hip center.

We conducted a retrospective study of the placement of porous-coated acetabular components using screws at more than 20 mm above the teardrop without structural bone graft for dysplastic hips to determine long-term outcome. Thirty hips (29 patients) were monitored for a mean of 15.2 years (range, 10.4-18.3 years) after surgery. Compared with 12 contralateral normal hips, the distance of the hip center from the teardrop was significantly high (26.8 +/- 4.8 and 13.4 +/- 2.7 mm P < .001); however, it was not laterally different (31. 5 +/- 5.1 and 31.7 +/- 5.0 mm). No acetabular components showed loosening. One metal shell was revised for wear and osteolysis. Morselized bone grafted in 25 hips was incorporated in all cases. Slight elevation of the hip center without lateralization in cementless cups fixed with screws was well tolerated for dysplastic hips.

Stem subsidence of polished and rough double-taper stems: in vitro mechanical effects on the cement-bone interface.

BACKGROUND AND PURPOSE: Many clinical reports have indicated that polished hip stems show better clinical results than rough stems of the same geometry. It is still unknown, however, what the mechanical effects are of different surface finishes on the cement at the cement-bone interface. We compared mechanical effects in an in vitro cemented hip arthroplasty model. METHODS: Two sizes of double-taper polished stems and matt-processed polished stems (rough stems) were fixed into composite femurs. A 1-Hz dynamic load was applied to the stems for 1 million cycles. An 8-h no-load period was set after every 16 h of load. Stem subsidence within the cement, and compressive force and horizontal cement creep at the cement-bone interface, were measured. RESULTS: Compared to rough stems, stem subsidence, compressive force and cement creep for polished stems were a maximum of 4, 12, and 7-fold greater, respectively. There was a strong positive correlation between stem subsidence and compressive force for polished stems. In contrast, a strong negative correlation was found between stem subsidence and compressive force for rough stems. There was also a statistically significant relationship between compressive force on the cement and cement creep for the polished stems, but no significant relationship was found for rough stems. INTERPRETATION: This is the first evidence that different surface finishes of stems can have different mechanical effects on the cement at the cement-bone interface. Stem subsidence in polished stems resulted in compressive force on the cement and cement creep. The mechanical effects that polished taper stems impart on cement at the cement-bone interface probably contribute to their good long-term fixation and excellent clinical outcome.

The relationship between stem subsidence and improvement in the radiolucency in polished tapered stems.

We studied the relationship between stem subsidence and improvement in the initial radiolucency at the bone-cement interface in polished- and rough-surface stems. Stem subsidence was seen in 34 of 42 hips (81.0%), and improvement in the initial radiolucency was seen in 15 hips (35.7%) in collarless polished tapered stems at 1 year after operation. In polished stems, stem subsidence in cases with improvement in the initial radiolucency was significantly greater than in those with no change (average 1.12 mm and 0.48 mm, P<0.005). In rough-surface stems partially coated with polymethylmethacrylate (VerSys Cemented Plus), no stem subsidence and no improvement in the initial radiolucency was seen. In polished stems, stem subsidence leads to cement creep and compressive force on the bone-cement interface and can improve the initial radiolucency.