Targeted Inactivation of p12Cdk2ap1, CDK2 Associating Protein 1, Leads to Early Embryonic Lethality

Targeted disruption of murine Cdk2ap1, an inhibitor of CDK2 function and hence G1/S transition, results in the embryonic lethality with a high penetration rate. Detailed timed pregnancy analysis of embryos showed that the lethality occurred between embryonic day 3.5 pc and 5.5 pc, a period of implantation and early development of implanted embryos. Two homozygous knockout mice that survived to term showed identical craniofacial defect, including a short snout and a round forehead. Examination of craniofacial morphology by measuring Snout Length (SL) vs. Face Width (FW) showed that the Cdk2ap1+/− mice were born with a reduced SL/FW ratio compared to the Cdk2ap1+/+ and the reduction was more pronounced in Cdk2ap1−/− mice. A transgenic rescue of the lethality was attempted by crossing Cdk2ap1+/− animals with K14-Cdk2ap1 transgenic mice. Resulting Cdk2ap1+/−:K14-Cdk2ap1 transgenic mice showed an improved incidence of full term animals (16.7% from 0.5%) on a Cdk2ap1−/− background. Transgenic expression of Cdk2ap1 in Cdk2ap1−/−:K14-Cdk2ap1 animals restored SL/FW ratio to the level of Cdk2ap1+/−:K14-Cdk2ap1 mice, but not to that of the Cdk2ap1+/+:K14-Cdk2ap1 mice. Teratoma formation analysis using mESCs showed an abrogated in vivo pluripotency of Cdk2ap1−/− mESCs towards a restricted mesoderm lineage specification. This study demonstrates that Cdk2ap1 plays an essential role in the early stage of embryogenesis and has a potential role during craniofacial morphogenesis

A size and shape analysis in obstructive sleep apnea patients

Obstructive Sleep Apnea (OSA) is related to an abnormal configuration of the upper airway. Since it presupposes a complex pathogenesis, studies on the disease usually require the analysis of many variables. This makes it difficult to obtain an adequate sample size. Further, the size of the upper airway structure often overwhelms the data matrix, and thus may be a source of multicollinearity or noise. More seriously, the strong size effect may hide an underlying biological factor. Landmark data and their analytic tools were employed in this study to partial out the size factor, and to decompose shape changes into uniform and non-uniform components. The non-uniform deformation was quantified in terms of bending energy by the Thin-Plate (TP) spline analysis. The Partial Least Square (PLS) method was applied to summarize the intricate data structure. The tongue was the unique upper airway structure for which size presented a significant association with OSA severity. In accordance with symptom severity, the hyoid bone and the submental region moved inferiorly and the fourth vertebra moved posteriorly with respect to the mandibular plane. This caused a fan-like configuration of the lower part of the upper airway in upright and supine body positions. Body position changes generated significant tongue deformation. TP splines revealed that the distinct tongue deformation caused by a body position change enable one to distinguish the asymptomatic group from the OSA subjects. Pharyngeal length was found to be proportionally associated with OSA symptoms. Results from the PLS analysis confirmed that the pharynx variables obtained in the upright position may best predict symptom severity. Overall, the new morphometric tools adopted here were found to be viable inOSA analysis.Dentistry, Faculty ofOral Biological and Medical Sciences (OBMS), Department ofGraduat

A comparative study of the relationship between airway size, tongue activity and body position

Airway obstruction in Obstructive Sleep Apnea (OSA) patients is believed to occur in the supine position during sleep. In order to investigate the relationship between upper airway size and genioglossus(GG) muscle activity, up-right (in natural head posture) and supine cephalograms were obtained for twenty OSA and ten asymptomatic control subjects. Tongue EMG and pressure recordings were obtained with the surface electrodes and pressure transducers in ten asymptomatic control subjects. The Student's t test and Wilcoxon signed rank test were used to test for differences between the two groups and between body positions. The OSA group revealed a longer tongue (p< 5%), a larger soft palate (p< 1%), an anteroposteriorly narrower and vertically lengthened upper airway (p< 1%), a inferiorly positioned hyoid bone (p< 1%), a more extended head posture (p< 5%) and a smaller hypopharynx (p< 1%) in the up-right standing position. After changing from the up-right to the supine position, the tongue cross-sectional area increased 4.3% (p< 5%) and oropharyngeal area decreased 3 6.5% (p< 1%) in the OSA group. When comparing the supine to the up-right control cephalograms, changes in tongue area were not observed, but the thickness of the soft palate increased (p< 1%). Differences in tongue cross- sectional area between two groups become significant with body positional changes from the up-right to the supine (p< 1%). With body positional changes, the hyoid bone moves superiorly toward the mandibular plane in the control group (p< 1%), but anteriorly toward the mandibular symphysis in the OSA group (p< 5%). The rest EMG activity of the GG muscle increased 33.8% (p< 5%) and the posterior tongue pressure increased 17% (p< 5%) with body positional changes from up-right to supine. Overall, the orophayngeal cross-sectional area collapsed 28.8% (p< 1%) despite a 34% increase (p< 5%) in GG muscle activity in the asymptomatic control group as a result of body positional changes. Furthermore, a 17% increase of tongue pressure on the posterior load cell indicates positional change of the tongue. In conclusion, it may not be the size of the soft palate alone but also the vertical and anteroposterior position of the tongue which could actively contribute to the development of OSA. Quantification of subtle differences in tongue and oropharyngeal size and position, geometry of the hyoid bone, upper airway muscle activity and tongue pressure accompanied by body positional changes aids in our understanding of the pathogenesis of OSA.Dentistry, Faculty ofGraduat

Potential Mechanisms Underlying Hypoxia-Induced Diabetes in a Rodent Model: Implications for COVID-19

Previous studies reported that repetitive hypoxia in rat pups reduces insulin secretion and elevates fasting blood glucose levels; these sequelae persisted for several months. This report describes how episodic hypoxic events elevate a chloride ion exporter, K+-Cl&minus; cotransporter-2 (KCC2), in the plasma membrane of insulin-secreting pancreatic &beta;-cells. We assume that acute diabetic symptoms observed in rat pups with periodic oxygen desaturation could result from a lack of blood insulin levels due to disturbed &beta;-cell function. This acute hypo-insulinemia may result from a disruption in chloride balance in &beta;-cells arising from an imbalanced KCC2-NKCC1 (chloride exporter-importer) density as a consequence of periodic oxygen desaturation. Mechanistically, we postulate that a reduced insulin secretion due to the KCC2-NKCC1 imbalance subsequent to acute oxygen desaturation could result in hyperglycemia in rat pups, paralleling symptoms shown in patients with COVID-19 who experienced acute respiratory distress

Disturbed zinc and ZIP8 homeostasis.

<p>(A) Western blot assay results on the whole cells harvested from both control and IH animals. ZIP8 protein concentration shows a noted decrease in the IH challenged group. When fractionated into cytoplasm and plasma membrane portions, (B) ZIP8 protein is expressed in the plasma membrane more than the cytoplasm portion. The IH group shows a lower density of ZIP8 in both cytoplasm and membrane. (C) Confocal images on beta cells demonstrate that ZIP8 transporters along the plasma membrane (indicated by pan-Cadherine protein resides in the membrane) markedly decreased after IH challenge. The bottom panel (D) shows that insulin expression also significantly decreased in the IH group. Scale bars indicate 5 µm. (E and F) Zinc taken up by the beta cells decreased in the IH group, yet not as much as the amount decreased in the siZIP8 treated cells (F). SCR indicates scrambled.</p

Comparisons on insulin and C-peptide concentrations between secreted amounts vs. produced amounts.

<p>The produced insulin (B) and C-peptide (D) measured in whole cell lysates do not show a difference; however, the secreted amounts obtained from each medium are significantly lower in the IH group when compared to control (A and C).</p

Serum levels of insulin (A) and C-peptide (B).

<p>Both insulin and C-peptide levels in serum of the treated animals are significantly lower than those of controls.</p

mRNA levels of <i>ZIP8</i> and <i>Ins1</i>.

<p>The mRNA level of <i>ZIP8</i> in the beta cells obtained from IH animals significantly lower than that of controls (A). The level of mRNA of the insulin gene <i>Ins1</i> remains the same (B).</p

Weights of dry mandibles that show a significant difference between control and IH animals at 5 weeks.

Weights of dry mandibles that show a significant difference between control and IH animals at 5 weeks.</p

Linear measurements estimate of the size of the maxilla and the mandible of rats.

A. Transverse view of the maxilla and the skull base: Intermolar width–distance between upper buccal cusp tips of the maxillary first molars; Palatal width–linear distance between the right and left interdental alveolar bone of the first and the second upper molars; Inter-Zygoma width–distance between the most posterior curvature of the zygoma; Maxilla length–distance between the middle of interdental alveolar bones of the upper central teeth and posterior nasal spine; B. Sagittal view of the mandible from approximately 45 degrees anterior and superior: Intercondylar width–distance connecting the midpoint of the right and left condylar heads; Mandible length–distance between the midpoint of the condylar head and interdental bone of the lower central incisors; Mandible height–longest distance between the lower and upper border of the ramus.</p