Perceived Anxiety in Family Caregivers of Individuals with Autism Spectrum Disorder, Down Syndrome and Williams Syndrome during the Lockdown of the First COVID-19 Wave in Spain.

The lockdown during the first wave of COV- ID-19 in Spain has been related to higher levels of anxiety in the general population. However, the emotional impact on Spanish caregivers of individuals with neurodevelopmental disorders (NDD) has not been studied so far.

Ansiedad percibida en familiares cuidadores de personas con trastorno del espectro autista, síndrome de Down y síndrome de Williams durante el confinamiento de la primera ola por COVID-19 en España / perceived anxiety in family caregivers of individuals with autism spectrum disorder, down syndrome and williams syndrome during the lockdown of the first COVID-19 wave in Spain

Antecedentes. El confinamiento durante la primera oleada de COVID-19 en España se ha relacionado con niveles mayores de ansiedad en la población general. Sin embargo, no se ha estudiado el impacto emocional en los cuidadores de personas con trastornos del neurodesarrollo (TND).Método. Se distribuyó un cuestionario a las organizaciones que prestan apoyo a las personas con TND y sus familias. Se analizaron los datos de los cuidadores de personas con trastorno del espectro autista (TEA) (N=17), síndrome de Down (N=25) y síndrome de Williams (N=18).Resultados. Los cuidadores informaron de preocupaciones relacionadas con la situación de pandemia y confinamiento. Los cuidadores de personas con TEA mostraron mayor preocupación sobre los conflictos familiares. Los tres grupos informaron de niveles de ansiedad más altos durante el confinamiento. Predijeron la ansiedad el trastorno de ansiedad previo y el diagnóstico del hijo/a. Conclusiones. Los predictores de ansiedad en los cuidadores de individuos con TND difieren de los reportados previamente en la población general española. Los resultados sugieren que el confinamiento fue especialmente duro paralas familias de personas con TEA. Las políticas públicas deberían considerar las necesidades de las personas con TND y sus cuidadores para minimizar las consecuencias negativas de la pandemia. (AU)

Background. The lockdown during the first wave of COVID-19 in Spain has been related to higher levels of anxietyin the general population. However, the emotional impact on Spanish caregivers of individuals with neurodevelopmental disorders (NDD) has not been studied so far. Methods. An online questionnaire was distributed to Spanish organisations providing support to individuals with NDD and their families. Data from caregivers of individuals with autism spectrum disorder (ASD) (N = 17), Down syndrome (DS)(N = 25) and Williams syndrome (WS) (N = 18) were analysed. Results. All caregivers reported concerns directly related to the pandemic and lockdown situation. Caregivers of individuals with ASD showed higher level of concern about the possibility of family conflict. All three groups reported higher levels of anxiety during the lockdown. Anxiety was predicted by previous anxiety disorder and the child’s diagnosis. Conclusions. Predictors of anxiety in caregivers of individuals with NDD differ from those previously reported in the general Spanish population. The results suggest that confinement in Spain was especially demanding for families of individuals with ASD. Public policies should consider the particular needs of people with NND and their caregivers to minimise the negative consequences of the ongoing pandemic.(AU)

Anxiety and Worries of Individuals with Down Syndrome During the COVID-19 Pandemic: A Comparative Study in the UK.

The present study explored the effects of the pandemic on individuals with Down Syndrome (DS; n = 67) compared to other groups with Special Education Needs and Disabilities (SEND; n = 48) and their Typically Developing Siblings (TDS; n = 56). In total, 115 caregivers reported on their own anxiety and worries and of their children. Anxiety levels for individuals with DS appeared to be lower compared to other SEND populations and to TDS. In terms of worries, individuals with DS worried more about social-related worries but worried less about family-related aspects compared to the other groups. In sum, individuals with DS might show less anxiety but still worried more about specific aspects related to the impact of COVID-19 pandemic on their lives.

The Impact of COVID-19 on Anxiety and Worries for Families of Individuals with Special Education Needs and Disabilities in the UK.

COVID-19 has affected people across the world. The current study examined anxiety and worries during the first UK national lockdown in March 2020. Parents (n = 402) reported on their own anxiety and worries as well as that of their son/daughter with Special Education Needs and Disabilities (SEND) and typically developing (TD) child (n = 186) at three time points. Although both groups showed increased anxiety across the three time points, levels of anxiety in the SEND group, but not the TD siblings, were predicted by awareness about COVID-19. In addition, worries differed between the groups showing that COVID-19 impacts the wellbeing of those with SEND differently to that of their TD siblings.

Nucleotide sequence of the 5'-terminus of Newcastle disease virus and assembly of the complete genomic sequence: agreement with the "rule of six".

We have determined the sequences of the 5' ends of three strains of Newcastle disease virus, permitting the assembly of the entire genomic sequence, which amounts to 15,186 nucleotides. This length is in agreement with the rule of six, which has been shown to determine replication efficiency in similar viruses. Comparison of the extreme 5' end of the trailer sequence with that of the 3'-terminal leader sequence of the virus reveals a high degree of complementarity. Variation between the 5'-terminal sequences of the different strains reveals the presence of alternative L gene polyadenylation signals, leading to correspondingly different trailer lengths.

Zn2+-sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal-induced protein-DNA dissociation.

SmtB is a member of a family of repressors which dissociate from DNA in the presence of metals; Zn2+ being the most potent inducer of metallothionein gene (smtA) transcription in vivo. In Synechococcus PCC 7942 cells devoid of chromosomal smtB, four plasmid-encoded mutants of SmtB (C61S, T11S/C14S, C121S and H105R/H106R) repressed lacZ expression driven by the smtA operator-promoter. Gel retardation assays with extracts from the complemented cells detected multiple SmtB-dependent complexes similar to those obtained with extracts from wild-type cells or with recombinant-SmtB. Elevated [Zn2+] alleviated repression in vivo by all of the mutants except H105R/H106R. These His residues (one or both) are therefore essential for Zn2+-sensing while, contrary to expectations, Cys residues are not. Hence different motifs facilitate metal-induced DNA-dissociation by SmtB and ArsR (the related oxyanion-sensing repressor), presumably generating variety in the spectra of metals sensed. Nucleotides and amino acids involved in DNA-SmtB interaction have been further defined/inferred and we also confirm that additional unknown factors form specific associations with the smt operator-promoter in elevated [Zn2+].

The Newcastle disease virus V protein binds zinc.

The V protein of Newcastle disease virus (NDV) is produced by the insertion of a single nontemplated G residue at a specific point during transcription of the phosphoprotein (P) gene, accessing a new reading frame upon translation. The V protein, in common with its counterpart in other paramyxoviruses contains a highly cysteine rich motif near the carboxyl terminus, suggestive of a zinc-binding domain. By constructing E. coli overexpression plasmids for the NDV P and V proteins, and monitoring the binding of 65ZnCl2 to proteins electroblotted onto nitrocellulose membranes, we have demonstrated that the V protein strongly binds zinc.

The 36K polypeptide synthesized in Newcastle disease virus-infected cells possesses properties predicted for the hypothesized 'V' protein.

Newcastle disease virus (NDV) virions possess two proteins which react strongly with monoclonal antibody 688 following separation by high resolution two-dimensional (isoelectric focusing/SDS) PAGE and detection by Western blotting. One is the phosphorylated nucleocapsid-associated 53K [P (NAP)] protein, the other comigrates with the 36K protein detected by radiolabelling NDV-infected chick embryo fibroblasts. [35S]Cysteine/[3H]leucine dual-labelling experiments show that the 36K protein is very rich in cysteine compared to the P (NAP) protein. In the Beaudette C strain it comigrates on one-dimensional SDS-polyacrylamide gels with the matrix protein (M); however, it is resolved from the slower migrating M protein from the Ulster strain of NDV. The size, strain-specific isoelectric point, high cysteine content and antigenic relatedness to the P (NAP) protein suggest that the 36K protein is the 'V' protein of NDV, the counterpart of which is found in other Paramyxoviridae.

A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV.

The hemagglutinin-neuraminidase (HN) gene from the Beaudette C strain of Newcastle disease virus (NDV) has been expressed in a recombinant fowlpox virus vector. The HN gene, under the control of the vaccinia p7.5 promoter, was inserted into a nonessential gene in the terminal inverted repeats of fowlpox virus. Expression was demonstrated in tissue culture, a protein of the correct size for fully glycosylated HN protein being recognized by an HN-specific monoclonal antibody on Western blots. When the recombinant fowlpox virus was inoculated into chickens by intravenous or wing-web routes, antibody which recognizes HN from purified NDV virions was produced. Protective immunity to NDV was generated in the chickens; at the highest dose of vaccine 100% of the chickens tested were protected against challenge with a virulent strain of NDV.

A fowlpox virus vaccine vector with insertion sites in the terminal repeats: demonstration of its efficacy using the fusion gene of Newcastle disease virus.

In this paper we report the development and testing of a fowlpox virus vector system. Insertion sites in non-essential regions within the terminal inverted repeats of the virus have been characterised. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus (NDV) has been inserted into a vaccine strain of fowlpox virus, and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of NDV and to elicit the formation of anti-fusion protein antibody.

Insertion of the fusion gene from Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant.

In this paper we report on the identification of non-essential genes in the terminal repeats of the avipox-virus fowlpox virus and the use of these as insertion sites in a vector system. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus has been inserted into a vaccine strain of fowlpox virus and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of Newcastle disease virus and to elicit the formation of an anti-fusion protein antibody.

Location of neutralizing epitopes on the fusion protein of Newcastle disease virus strain Beaudette C.

A panel of eight neutralizing monoclonal antibodies (MAbs) against the fusion (F) protein of Newcastle disease virus (NDV) has been shown to locate a major antigenic site on the basis of competitive binding assay and additivity index studies. Five epitopes (A1 to A5) have been located within this site on the F protein of the Beaudette C strain of NDV on the basis of cross-resistance plaque assays of MAb-resistant mutants raised against these MAbs. Epitopes A1, A4 and A5 are distinct; epitope A2 partially overlaps epitope A3. Nucleotide sequence analysis of the F genes of MAb-resistant mutants showed that each predicted single amino acid substitutions ranging from amino acid residues 157 to 171 for epitope A4 and at residues 72, 78, 79 and 343 for epitopes A1, A2, A3 and A5 respectively. These locations indicate that both the F1 and F2 fragments are involved in the formation of a single antigenic site and suggest the involvement of extensive protein folding in the active form of this F protein.

Effect of amino acid substitutions on glycosylation of the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus strain Beaudette C.

The nucleotide sequences of two monoclonal antibody-resistant mutant viruses predict changes from the wild-type in the number of potential glycosylation (Asn-X-Thr/Ser) in the mutant haemagglutinin-neuraminidase (HN) glycoproteins of the Beaudette C strain of Newcastle disease virus. The HN glycoproteins of these mutants, F5 and Z18, migrate either slower (F5) or faster (Z18) than that of the wild-type in SDS-PAGE. HN proteins synthesized in chick embryo fibroblasts following infection by either mutant or wild-type virus in the presence of tunicamycin (an inhibitor of glycosylation), comigrate on SDS-PAGE. These results confirm that the HN protein of the mutant virus, F5, has gained a glycosylation site at Asn(323)-Ser-Ser and that the conserved potential glycosylation site at Asn(481)-His-Thr is indeed glycosylated in the HN protein of the wild-type Beaudette C strain of Newcastle disease virus but is lost in that of the mutant virus, Z18.

Mapping of three antigenic sites on the haemagglutinin-neuraminidase protein of Newcastle disease virus.

Nine neutralizing monoclonal antibodies (MAbs), each of which react with the haemagglutinin-neuraminidase (HN) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV), have been used in competitive binding assays to delineate three non-overlapping antigenic sites A, B and C. Epitopes within these sites have been identified on the basis of cross-reactivity of MAb-resistant mutants against the panel of MAbs, determined by plaque assays and Western blotting. Site A contains three non-overlapping epitopes (A1, A2 and A3). A1 is the only linear epitope; all remaining epitopes are conformational. MAbs which react with epitopes A2 and A3 inhibit neuraminidase activity (NA) when assayed with neuraminlactose. Site B contains three partially overlapping epitopes (B1, B2 and B3) and site C is represented by a single epitope (C1). HN gene sequence analysis of MAb-resistant mutants showed that they each had only single amino acid substitutions which range from amino acid residues 347-460 for site A, 284-325 for site B, and at 481 for the C1 epitope. The apparent molecular mass of the HN glycoprotein of one mutant was increased from 72 to 75 kDa. This correlates well with the creation of an additional potential glycosylation site in this mutant from Asn-Ser-Pro(325) to Asn-Ser-Ser(325).

Location of a neutralizing epitope for the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the alpha-peptide of beta-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides.

The purification of four respiratory syncytial virus proteins and their evaluation as protective agents against experimental infection in BALB/c mice.

The fusion (F) glycoprotein, large glyco- (G) protein, phospho- (P) protein and 22K protein of respiratory syncytial (RS) virus A2 strain were purified by a combination of immunoaffinity adsorption and preparative SDS-PAGE. All four proteins elicited serum antibody in mice after repeated inoculation in adjuvant, although the magnitude of the response as measured by ELISA varied from mouse to mouse. The F protein generated neutralizing antibodies in only 50% of the mice determined to be seropositive by ELISA. The G protein also induced neutralizing antibodies but in this instance neutralization tests and ELISA titres were more closely correlated. No neutralizing activity was detected in mice immunized with the P or 22K proteins although all produced antibody detectable by ELISA. Mice immunized with either the F or the G protein were found to be protected against subsequent RS virus challenge, whether they had developed neutralizing antibody or not. Mice inoculated with the P or 22K proteins were not protected.

Identification of haemagglutinin-neuraminidase antibody binding sites by Western blot analysis of antibody-resistant mutants and partial digest fragments of Newcastle disease virus.

A collection of monoclonal antibodies (MAbs) which react with the haemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) has been used to isolate MAb-resistant mutants of the Beaudette C strain of NDV. The patterns of cross-reactivity of the HN proteins of these mutants against the collection of MAbs determined by Western blotting allowed the MAbs to be sorted into different groups. Protease V8 partial digest fragments of purified wild-type virions and subsequent reaction against the collection of MAbs using Western blotting provided an alternative method of grouping MAbs which broadly agreed with the former method. Chemical cleavage of the HN protein at aspartate-proline bonds followed by Western blotting of the fragments allowed the approximate position of certain MAb binding sites to be determined.

NCL-5D3: a new monoclonal antibody recognizing low molecular weight cytokeratins effective for immunohistochemistry using fixed paraffin-embedded tissue.

Production of a new monoclonal antibody designated NCL-5D3 is described. The antibody recognizes several low molecular weight cytokeratins, in particular cytokeratin Moll number 8 as determined by immunoblotting studies, and is highly effective for immunocytochemistry using routinely processed paraffin-embedded material. Staining is enhanced by prior treatment of the sections with trypsin. Assessment using a wide variety of normal and neoplastic tissue indicates reactivity with all tissues of simple or glandular epithelial origin, and in addition with many squamous carcinomas. Thus the antibody should prove of value in diagnostic histopathology.

Strain variation of respiratory syncytial virus.

Western blotting and immunoperoxidase staining with monoclonal antibodies were employed to analyse epitopic and polypeptide molecular weight variation among isolates of respiratory syncytial (RS) virus collected in Newcastle between 1965 and 1986. One group of isolates resembled the A2 and Long prototype subgroup A strains of RS virus in possessing a P protein of Mr 34,000. Isolates in this subgroup showed two patterns of reactivity with subgroup A-specific monoclonal antibodies to the G glycoprotein and 22K protein. Isolates with both reactivity patterns were isolated throughout the period studied. Isolates in the second group resembled the 8/60 subgroup B prototype strain in their lack of reactivity to subgroup A-specific monoclonal antibodies but were heterogeneous in P protein molecular weight. The earliest isolate only, made in 1965, possessed a P protein of Mr 31,000 resembling the prototype strain. All subsequent subgroup B isolates possessed a higher Mr, 33,000, P protein. Overall, subgroup A viruses were isolated most frequently although subgroup B strains may have predominated in some epidemics.