Did we turn a blind eye? The answer is simply there. Peripheral pulmonary vascular thrombosis in COVID-19 patients explains sudden worsening of clinical conditions

COVID-19 patients suffering sudden worsening of clinical conditions have an atypical peripheral pulmonary arterial obstruction at computed tomography pulmonary angiogram (CTPA), poorly associated to deep venous thrombosis (DVT), suspicious for thrombotic in situ nature rather than embolic

The telomeric protein AKTIP interacts with A- and B-type lamins and is involved in regulation of cellular senescence

AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence

AKTIP/Ft1, a new shelterin-interacting factor required for telomere maintenance

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (humanAKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factoridentified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere disfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication

Neurocognitive and cerebellar function in ADHD, autism and spinocerebellar ataxia

The cerebellum plays a major role in balance, motor control and sensorimotor integration, but also in cognition, language, and emotional regulation. Several neuropsychiatric disorders such as attention deficit-hyperactivity disorder (ADHD), autism spectrum disorder (ASD), as well as neurological diseases such as spinocerebellar ataxia type 3 (SCA3) are associated with differences in cerebellar function. Morphological abnormalities in different cerebellar subregions produce distinct behavioral symptoms related to the functional disruption of specific cerebro-cerebellar circuits. The specific contribution of the cerebellum to typical development may therefore involve the optimization of the structure and function of cerebro-cerebellar circuits underlying skill acquisition in multiple domains. Here, we review cerebellar structural and functional differences between healthy and patients with ADHD, ASD, and SCA3, and explore how disruption of cerebellar networks affects the neurocognitive functions in these conditions. We discuss how cerebellar computations contribute to performance on cognitive and motor tasks and how cerebellar signals are interfaced with signals from other brain regions during normal and dysfunctional behavior. We conclude that the cerebellum plays a role in many cognitive functions. Still, more clinical studies with the support of neuroimaging are needed to clarify the cerebellum's role in normal and dysfunctional behavior and cognitive functioning

The ability to maintain rhythm is predictive of ADHD diagnosis and profile

Attention deficit hyperactivity disorder (ADHD) is the most prevalent neuropsychiatric disorder in the world. Currently, the diagnosis is based mainly on interviews, resulting in uncertainties in the clinical assessment. While some neuropsychological tests are used, their specificity and selectivity are low, and more reliable biomarkers are desirable. Previous research indicates that ADHD is associated with morphological changes in the cerebellum, which is essential for motor ability and timing. Here, we compared 29 children diagnosed with ADHD to 96 age-matched controls on prism adaptation, eyeblink conditioning, and timed motor performance in a finger tapping task. Prism adaptation and timing precision in the finger tapping task, but not performance on eyeblink conditioning, differed between the ADHD and control groups, as well as between children with and without Deficits in Attention, Motor control, and Perception (DAMP) – a more severe form of ADHD. The results suggest finger tapping can be used as a cheap, objective, and unbiased biomarker to complement current diagnostic procedures

AKTIP downregulation impairs telomere replication.

<p>Ctr or shAKTIP HeLa cells were synchronized at the G1/S boundary with a double thymidine block and harvested at the indicated times. (A) Scatter plots showing the proportions of cells in S phase in asynchronous cultures (As) and in cultures analyzed at various times after release from the double thymidine block. Prior to harvest at each time point, the cells were incubated with BrdU for 30 min. (B) ChIP analysis on synchronized HeLa cells incubated with BrdU for 1 h before harvesting. Precipitations were performed with an anti-TRF1 antibody. IgG antibody was used as negative control. Inputs represent 10 and 1% of genomic DNA. Dot-blot analysis was performed using telomeric or ALU repeat-specific probes. Precipitated DNA was analyzed by Western blotting with an anti-BrdU antibody. (C, D) Quantification of the data expressed in arbitrary units (A.U.) of unlabeled (C) or BrdU-labeled (D) precipitated telomeric DNA at the different time points of analysis, each normalized to input samples. The graphs show three independent experiments, with error bars indicating the SD.</p

AKTIP downregulation impairs DNA replication.

<p>(A) FACS analysis of 10 dpi ctr and shAKTIP-11 HPFs incubated with BrdU for 30 min, fixed, and then stained for BrdU and DNA (with PI). AKTIP depletion results in an S phase block; percents of cells in different cell cycle phases are reported in the upper right corner of each panel. (B, C) PCNA localization in unsynchronized mock, 10 dpi ctr, 10 dpi shAKTIP-11, HU-treated and APC-treated HPFs. Examples (B) and quantification of PCNA positive nuclei (C) from unextracted or Triton X-100-extracted HPFs. Bars are the mean values ± SD of samples analyzed in duplicate. ** and * indicate significant difference from control with p<0.01 and p<0.05 in the Student t test. (D, E) Distribution of nuclei with different S phase PCNA staining patterns. Bars in the graph (E) are the mean values ± SD of samples analyzed in duplicate; colours in E are as in the representative images shown in D; distributions of ctr and shAKTIP nuclei are significantly different in the Student t test with p<0.05. (F) AKTIP-GST pulls down PCNA and RPA70 from 293T cell extracts.</p

AKTIP interacts with telomeric DNA, TRF1 and TRF2.

<p>(A) ChIPs from HPFs, uninfected HeLa cells and shAKTIP-11-infected HeLa cells reveal interactions between AKTIP and telomeric DNA. Chromatin was immunoprecipitated with an anti-AKTIP antibody or control IgGs; slot-blots were hybridized with TTAGGG or ALU repeat probes. (B) ChIP quantification after normalization to the input (levels shown in A). Bars show the mean values of two experiments ± SD; the amount of telomeric DNA precipitated from uninfected HeLa cells is significantly higher than that obtained from shAKTIP-11-infected cells (*p<0.05 in the Student t test). (C, D) AKTIP-GST pulls down TRF1 (C) and TRF2 (D) from 293T cell extracts. In, input; M, MW markers.</p

AKTIP directly binds TRF1 and TRF2.

<p>(A) A tridimensional molecular model for AKTIP. The arrows point to the starting sites of the disordered N- and C-terminal regions (not depicted); the variant Asp residue and His-Pro-Leu motif are represented as sticks and indicated by red and purple arrows, respectively (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005167#pgen.1005167.s005" target="_blank">S5 Fig</a>). (B) Schematic organization of the AKTIP protein; the AKTIP truncations used for GST pulldown are indicated below the scheme. (C, D) In vitro mapping the AKTIP regions that interact with TRF1 or TRF2 using bacterially purified proteins; the UEV domain of AKTIP binds both TRF1 and TRF2.</p

AKTIP depletion affects cell cycle progression and induces the DNA damage response (DDR).

<p>(A) Relative mitotic indexes (± SD) of shAKTIP (hairpin sequences 9, 11 and 13) and control (ctr) at 10 dpi. (B) Immunoblotting of 10 dpi extracts from shAKTIP HPFs shows accumulation of cyclins compared to ctr; in the graph, blot signals are normalized relative to the actin used as a loading control. (C) Cumulative population doublings of HPFs, HeLa and 293T cells transduced with ctr or shAKTIP-11. (D) Immunoblots of 10 dpi extracts reveal DNA damage signaling in shAKTIP samples. Densitometric analysis showed that in 9, 11 and 13 RNAi cells there is a 2.7-, 2.6-, and 3.3-fold increase of Chk1, respectively; in the same cells ATM-P Ser 1981 increases were 1.4-, 1.6- and 2.0-fold, respectively. (E) Q-PCR of total RNA shows a strong increase in p21 expression in shAKTIP-11 HPFs relative to control; samples collected at 7 dpi were analyzed in duplicate and shown as mean values ± SD. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005167#pgen.1005167.s001" target="_blank">S1 Fig</a>.</p