FAM122A ensures cell cycle interphase progression and checkpoint control by inhibiting B55α/PP2A through helical motifs.

The Ser/Thr protein phosphatase 2 A (PP2A) regulates the dephosphorylation of many phosphoproteins. Substrate recognition are mediated by B regulatory subunits. Here, we report the identification of a substrate conserved motif [RK]-V-x-x-[VI]-R in FAM122A, an inhibitor of B55α/PP2A. This motif is necessary for FAM122A binding to B55α, and computational structure prediction suggests the motif, which is helical, blocks substrate docking to the same site. In this model, FAM122A also spatially constrains substrate access by occluding the catalytic subunit. Consistently, FAM122A functions as a competitive inhibitor as it prevents substrate binding and dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. FAM122A deficiency in human cell lines reduces the proliferation rate, cell cycle progression, and hinders G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells attenuates CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a short helical motif (SHeM)-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.

FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase.

The Ser/Thr protein phosphatase 2A (PP2A) is a highly conserved collection of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). PP2A/B55 substrate recruitment was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM [ RK ]- V -x-x-[ VI ]- R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A. This conserved SLiM is necessary for FAM122A binding to B55α in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational and biochemical data and supports a mechanism whereby FAM122A uses the 'SLiM' in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, progression through cell cycle transitions and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a 'SLiM'-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.

Caregiver Burden in Adult Solid Organ Transplantation.

The informal caregiver plays a critical role in supporting patients with various end-stage diseases throughout the solid organ transplantation journey. Caregiver responsibilities include assistance with activities of daily living, medication management, implementation of highly specialized treatments, transportation to appointments and treatments, and health care coordination and navigation. The demanding nature of these tasks has profound impacts across multiple domains of the caregiver's life: physical, psychological, financial, logistical, and social. Few interventions targeting caregiver burden have been empirically evaluated, with the majority focused on education or mindfulness-based stress reduction techniques. Further research is urgently needed to develop and evaluate interventions to improve caregiver burden and outcomes for the patient-caregiver dyad.

Attenuation correction effects on SPECT/CT procedures: phantoms studies.

Attenuation correction is widely used in SPECT/CT (Single Photon Emission Computed Tomography) procedures, especially for imaging of the thorax region. Different compensation methods have been developed and introduced into clinical practice. Most of them use attenuation maps obtained using transmission scanning systems. However, this gives extra dose of radiation to the patient. The purpose of this study was to identify when attenuation correction is really important during SPECT/CT procedures.For this purpose, we used Jaszczak phantom and phantom with three line sources, filled with technetium ((99m)-Tc), with scattering materials, like air, water and acrylic, in different detectors configurations. In all images acquired were applied analytic and iterative reconstruction algorithms; the last one with or without attenuation correction. We analyzed parameters such as eccentricity, contrast and spatial resolution in the images.The best reconstruction algorithm on average was iterative, for images with 128 × 128 and 64 × 64 matrixes. The analytical algorithm was effective only to improve eccentricity in 64 × 64 matrix and matrix in contrast 128 × 128 with low statistics. Turning to the clinical routine examinations, on average, for 128 × 128 matrix and low statistics counting, the best algorithm was the iterative, without attenuation correction,improving in 150% the three parameters analyzed and, for the same matrix size, but with high statistical counting, iterative algorithm with attenuation correction was 25% better than that without correction. We can conclude that using the iterative algorithm with attenuation correction in the water, and its extra dose given, is not justified for the procedures of low statistic counting, being relevant only if the intention is to prioritize contrast in acquisitions with high statistic counting.

Digging the New York City Skyline: soil fungal communities in green roofs and city parks.

In urban environments, green roofs provide a number of benefits, including decreased urban heat island effects and reduced energy costs for buildings. However, little research has been done on the non-plant biota associated with green roofs, which likely affect their functionality. For the current study, we evaluated whether or not green roofs planted with two native plant communities in New York City functioned as habitats for soil fungal communities, and compared fungal communities in green roof growing media to soil microbial composition in five city parks, including Central Park and the High Line. Ten replicate roofs were sampled one year after planting; three of these roofs were more intensively sampled and compared to nearby city parks. Using Illumina sequencing of the fungal ITS region we found that green roofs supported a diverse fungal community, with numerous taxa belonging to fungal groups capable of surviving in disturbed and polluted habitats. Across roofs, there was significant biogeographical clustering of fungal communities, indicating that community assembly of roof microbes across the greater New York City area is locally variable. Green roof fungal communities were compositionally distinct from city parks and only 54% of the green roof taxa were also found in the park soils. Phospholipid fatty acid analysis revealed that park soils had greater microbial biomass and higher bacterial to fungal ratios than green roof substrates. City park soils were also more enriched with heavy metals, had lower pH, and lower quantities of total bases (Ca, K, and Mg) compared to green roof substrates. While fungal communities were compositionally distinct across green roofs, they did not differentiate by plant community. Together, these results suggest that fungi living in the growing medium of green roofs may be an underestimated component of these biotic systems functioning to support some of the valued ecological services of green roofs.

The protective effects of resveratrol and L-NAME on visceral organs following aortic clamping.

BACKGROUND: This study investigated the effect of temporary occlusion of the aorta on the development of ischemia-reperfusion (I/R) injury of the visceral organs, the optimal timing of administration of resveratrol, and its mechanism of protection via inhibiting nitric oxide (NO) release with an NO synthase inhibitor. METHODS: Rabbits were divided into seven groups according to the administration period of resveratrol and/or N(G)-nitro-L-arginine methyl ester (L-NAME): control group; group 1, resveratrol during ischemic period; group 2, resveratrol during reperfusion period; group 3, L-NAME during ischemic period; group 4, L-NAME during reperfusion period; group 5, resveratrol during ischemic period and L-NAME during reperfusion period; group 6, L-NAME during ischemic period and resveratrol during reperfusion period. The infrarenal aorta was clamped for 30 min. Blood samples were taken for the biochemical assessment, and organ specimens were taken for pathological assessment at 24hr of reperfusion. RESULTS: In groups 5 and 6, the renal I/R injury was comparatively milder (I/R injury score 1.04+/-0.29 in control group, 0.25+/-0.17 in group 5, and 0.33+/-0.13 in group 6 [p<0.05]). The I/R injury of bowel was milder in group 5 (I/R injury score 1.8+/-0.80 in control group vs. 0.0+/-0.0 in group 5 [p<0.05]). CONCLUSION: The protective effects of resveratrol on organs that have high metabolic rate like kidney and bowel was proven histopathologically. It may be beneficial to use different pharmacological medications in different periods of the I/R damage as they represent different characteristics with and without oxygen. The combination of resveratrol and L-NAME against I/R injury appears to be an effective option in the near future.

Collaborative therapy with nebivalol and L-NAME for spinal cord ischemia/reperfusion injury.

Postoperative neurologic deficit is the most devastating complication after thoracoabdominal aortic aneurysm repair. Our aim was to investigate whether nebivolol has protective effects during ischemia or reperfusion and the most effective mechanism of protection via inhibiting nitric oxide (NO) release with an NO synthase inhibitor in an experimental model of spinal cord ischemia/reperfusion injury. Spinal cord ischemia was induced by occlusion of the infrarenal aorta for 30 min. Thirty-one rabbits were divided into five groups according to the administration period of nebivolol and/or N(G)-nitro-L-arginine methyl ester (L-NAME): control group; group NI, nebivolol during ischemic period; group NR, nebivolol during reperfusion period; group NILR, nebivolol during ischemic period and L-NAME during reperfusion period; and group LINR, L-NAME during ischemic period and nebivolol during reperfusion period. Blood samples were taken at both ischemia and reperfusion periods to obtain nitrite/nitrate levels. After neurologic evaluation at 24 hr of reperfusion, malondialdehyde (MDA) levels were measured. Neurologic impairment was significantly lower in group LINR (Tarlov score 3.4 +/- 0.6, p < 0.05). MDA levels were lower in nebivolol-treated animals, but the lowest value was achieved in the NR group, 35.6 +/- 2.7 nmol/g (p < 0.001). Nitrite levels were decreased significantly in all nebivolol-treated animals in the reperfusion period, but the lowest value was measured in the LINR group (455 +/- 137 vs. 1,760 +/- 522 nmol/mL, p < 0.001). Prophylactic use of nebivolol reduced neurologic injury, and combining with L-NAME provided the best clinical improvement by attenuating the inflammatory mileu in this experimental model. Combination of nebivolol and L-NAME appears to be an effective option for spinal cord protection against ischemia/reperfusion injury.

Dobutamine stress echocardiography and left ventricular mass changes of mechanical aortic valve replacement in four years follow-up.

AIM: The aim of this study was to investigate the differences in cardiac response to stress according to the size of the prosthetic valve in patients who underwent aortic valve replacement (AVR) and to evaluate the relationship between the size of the prosthetic valve and cardiac recovery-remodeling after the operation. METHODS: Thirty patients who had undergone AVR (12 patients) or double valve replacement (18 patients) underwent dobutamine-stress echocardiography 4.2 years after the operation to evaluate response to stress . They were divided into 2 groups according to valve prosthesis size. The small-size AVR group (group 1, n=17) had prosthetic aortic valves 21 pounds mm; the large-size AVR group (group 2, n=13) had valves >21 mm. Response to stress and preoperative and postoperative echocardiographic findings were compared. Pulsed and continuous-wave Doppler studies were performed at rest and at the end of each stage. Peak and mean aortic gradients, left ventricular diastolic and systolic functions were measured for each group. RESULTS: Dobutamine stress increased heart rate and blood pressure in both groups. Peak pressure gradient across the aortic valve prostheses was 42.1 mm Hg in group 1 and 20.9 mm Hg in group 2 (P<0.05) at rest. After dobutamine infusion, the peak pressure gradient across the aortic valve prostheses increased to 85.1 mm Hg in group 1 and 54 mm Hg in group 2 (P<0.05). Isovolumetric relaxation time returned to normal in both groups following dobutamine infusion; this decrease was significant only in group 1. Patients achieved a decrease in left atrium and left ventricular diameters and volumes, as evidence of remodeling following AVR. Left ventricular mass index (LVMI) decreased from 127.6+/-47.6 to 98.1+/-36.9 and from 159.9+/-16.1 to 125.3+/-10.1 in groups 1 and 2, respectively, but this decline was not statistically significant. CONCLUSIONS: Smaller valves have higher gradients and this significant difference increases under stress. Significant improvement in echocardiographic diameters, cardiac filling volumes and LVMI reflects the benefit of the operation. Cardiac remodeling is independent of valve size, although high transprosthetic gradients occur during stress conditions.

Increased expression of mRNA encoding calbindin-D28K, the glucose-regulated proteins, or the 72 kDa heat-shock protein in three models of acute CNS injury.

Changes at the level of gene expression are becoming an increasingly recognized component of the neuronal response to injury. We used Northern analysis and three in vivo models of central nervous system (CNS) injury in the rat to determine whether injury alters the expression of certain gene products related to cellular homeostasis. The three models included kainate (KA)-induced seizures, global ischemia, and lateral fluid percussion injury to the cerebral cortex. Animals were sacrificed at various times after injury, and total RNA was isolated from specific brain regions. Northern blots were hybridized with probes for calbindin-D28K, the 78 and 94 kDa glucose-regulated proteins (grp78, grp94), the inducible 72 kDa heat-shock protein (hsp72), and a control probe for the 18S ribosomal subunit. Results showed that mRNA for calbindin-D28K, grp78, and hsp72 increased in the hippocampus following seizures. Peak expression occurred 6-12 h after administration of KA, and returned towards baseline in most cases by 24 h. Changes in all four transcripts were seen in the hippocampus or cortex following global ischemia, although the return to baseline tended to exceed 24 h for the grps. In the trauma model, mRNA for hsp72 was increased in the cortex ipsilateral to the impact 12 h after injury. These results expand the repertoire of known changes in mRNA expression following CNS injury. The increases in hsp72 and grps indicate the occurrence of a generalized stress response. Furthermore, given the evidence that grp78 and grp94 are induced by calcium ionophores in vitro, and the potential role of calbindin-D28K in buffering cytoplasmic calcium, the changes observed in this study may represent a cellular response to perturbed calcium homeostasis that is known to occur in acute CNS injury.

GM1 reduces infarct volume after focal cerebral ischemia.

The efficacy of monosialoganglioside GM1 treatment was evaluated in a model of experimental stroke. Cerebral ischemia was induced by permanent occlusion of left middle cerebral artery. GM1 was administered intravenously soon after the occlusion of the artery and then intramuscularly daily for 7 days. Results indicate that GM1 can reduce the extent of infarct volume and neurochemical deficits associated with the ischemic event. The protection was more evident in the cortex than in the caudate-putamen. These observations confirm and extend the evidence of the GM1 efficacy in experimental models of stroke and further support the usefulness of gangliosides in the treatment of these pathologies.

Orally administered glycolipid derivative LIGA20 reduces infarct volume and behavioral impairment after focal cerebral ischemia.

The efficacy of p.o. semisynthetic glycolipid LIGA20 (II3Neu5-AcGgOse4-2-d-erythro-1,3-dihydroxy-2-dichloro-aceta mide-4-trans- octadecene) treatment in stroke was studied in a permanent left middle cerebral artery occlusion model in the rat. A dose-dependent increase of plasma LIGA20 and its presence in the brain were documented after p.o. drug application. Oral administration of 50 to 200 mg/kg of LIGA20, initiated 24 hr before middle cerebral artery occlusion and continued for 7 days, reduced the motor and cognitive impairment after the stroke, measured by the rotarod and the passive avoidance test, respectively. The 10-mg/kg dose was effective when given i.v. but not p.o. Oral treatment with 100 mg/kg of LIGA20 reduced the infarct size in the cortex but not in the ischemic core (the striatum). No biochemical or behavioral adverse effects of LIGA20 treatment were observed. Further studies are needed to evaluate the full therapeutic potential of this compound.

Prolonged increases in neurotrophic activity associated with kainate-induced hippocampal synaptic reorganization.

Synaptic reorganization occurs in the hippocampus following various forms of seizure activity and injury, and may contribute to epileptogenesis. To address the hypothesis that neurotrophic factors play an inductive role in synaptic reorganization following seizures, we directly measured neurotrophic activity in rat hippocampal extracts after kainate injection or prolonged stimulation of the perforant path. Serial dilutions of hippocampal extracts were added to cultures of chick dorsal root ganglia, which are known to require trophic support from nerve growth factor and other neurotrophins, or ciliary ganglia neurons, which require trophic support from ciliary neurotrophic factor. Neurotrophic activity was significantly increased in hippocampal extracts harvested from 12 h to 2 months after kainate treatment, with the peak effect seen at seven days. This neurotrophic activity was substantially blocked by an anti-nerve growth factor antibody. Extracts at seven days also showed a significant increase in ciliary neurotrophic factor-like activity. Sulfide/silver histochemistry, which stains dentate granule cell axon terminals, revealed that mossy fiber sprouting was evident two weeks following kainate treatment and increased progressively over the next two to six weeks. Perforant path stimulation that produced hyperexcitability in the dentate gyrus, but no sprouting, failed to induce changes in neurotrophic activity. These results suggest there are significant increases in neurotrophic factors following kainate-induced seizures, and the increases may be related to kainate-induced hippocampal injury rather than seizures per se. Furthermore, the timecourse of increased neurotrophic activity parallels that of mossy fiber reorganization, and is consistent with the hypothesis that neurotrophic factors play a role in the injury-induced synaptic reorganization seen in epilepsy.

Time course, localization and pharmacological modulation of immediate early inducible genes, brain-derived neurotrophic factor and trkB messenger RNAs in the rat brain following photochemical stroke.

A focal, unilateral thrombotic stroke was produced in the rat sensorimotor cortex. The time course of expression and localization of the immediate early inducible genes: c-fos, c-jun, zif268; nerve growth factor, brain-derived neurotrophic factor and the related tyrosine kinase high-affinity receptor (trkB) messenger RNAs were studied by in situ hybridization. The levels of messenger RNAs for c-fos, zif268, brain-derived neurotrophic factor (but not nerve growth factor) and trkB were consistently increased in cortex ipsilaterally to the lesion, while c-jun messenger RNA content was only slightly increased. The brain-derived neurotrophic factor messenger RNA was increased from 2 to 18 h following the stroke, mainly in cells having a normal morphological appearance. The trkB messenger RNA displayed temporal and spatial increases similar to brain-derived neurotrophic factor messenger RNA. The time course and pattern of expression of immediate early inducible gene and trophic factor messenger RNAs did not clearly support a causal relationship between these two families of factors. The observed messenger RNA increases were greatly attenuated by the non-competitive N-methyl-D-aspartate-sensitive glutamate receptor antagonist (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine , but substantially unaffected by the non-N-methyl-D-aspartate receptor antagonist 2,3-dihydroxy-6-nitrosulphanoylbenzoquinoxaline. The results suggest a major contribution of N-methyl-D-aspartate-sensitive glutamate receptor activation to the transcriptionally directed events subsequent to stroke. Future studies should clarify the contribution of these processes to either the progression of neuronal degeneration or the establishment of protective compensatory responses.

Photochemical stroke and brain-derived neurotrophic factor (BDNF) mRNA expression.

In situ hybridization and Northern blotting were used to study the expression of brain-derived neurotrophic factor (BDNF) mRNA in the rat brain following photochemical stroke. A focal thrombotic lesion of the sensorimotor cortex was produced by intravenously injecting the light-sensitive dye rose bengal and exposing the skull to a controlled beam of light. Four hours after the light exposure the level of BDNF mRNA was increased in the hippocampus and cortex ipsilateral and perifocal to the lesion. The stroke-induced BDNF mRNA increase was prevented by the non-competitive glutamate receptor blocker dizocilpine (MK-801). The results indicate that the activation of N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors is involved in the stroke-triggered stimulation of BDNF mRNA increase.

Protective effects of a monosialoganglioside derivative following transitory forebrain ischemia in rats.

We evaluated the effects of treatment with the inner ester derivative of the monosialoganglioside GM1 on cortical electroencephalographic activity and hippocampal CA1 morphology after transitory, near-complete cerebral ischemia in rats. Ischemia was induced by the four-vessel occlusion method, and we studied only the 58 rats that showed flattening of the cortical electroencephalogram for the entire 30 minutes of occlusion. The ganglioside (n = 30) or saline (n = 28) was administered intravenously immediately after release of the carotid clips and then intramuscularly for 21 days of observation. Cortical electroencephalographic activity was monitored throughout the experiment. After 21 days of recirculation we assessed hippocampal CA1 damage by light microscopy. The results indicate that treatment with the ganglioside reduces postischemic secondary damage to the cortical circuitry (as indicated by significantly higher cortical electroencephalographic activity late after reperfusion) and limits neuronal loss in the CA1 region. Our results lend support to the possible therapeutic use of ganglioside in human pathologic conditions associated with cerebrovascular insufficiencies.

Hypoxic-ischemic damage and the neuroprotective effects of GM1 ganglioside.

In vitro studies have shown that monosialoganglioside GM1 reduces excitatory amino acid-related neurotoxicity by limiting the downstream consequences of abusive excitatory amino acid receptor stimulation. Systemic administration of GM1 appears to be efficacious in reducing acute neuronal damage and in facilitating medium- and long-term functional recovery after brain injury. We propose that GM1 protective effects in the acute injury phase results from attenuation of excitotoxicity, whereas the functional recovery seen at longer term could reflect GM1 potentiation of neuronotrophic factors. The potential therapeutic efficacy of GM1 administration in humans is suggested by clinical studies demonstrating improved neurologic outcome in stroke patients.