Cytokine release syndrome-associated encephalopathy in patients with COVID-19.

BACKGROUND AND PURPOSE: Neurological manifestations in coronavirus disease (COVID)-2019 may adversely affect clinical outcomes. Severe COVID-19 and uremia are risk factors for neurological complications. However, the lack of insight into their pathogenesis, particularly with respect to the role of the cytokine release syndrome (CRS), is currently hampering effective therapeutic interventions. The aims of this study were to describe the neurological manifestations of patients with COVID-19 and to gain pathophysiological insights with respect to CRS. METHODS: In this longitudinal study, we performed extensive clinical, laboratory and imaging phenotyping in five patients admitted to our renal unit. RESULTS: Neurological presentation included confusion, tremor, cerebellar ataxia, behavioral alterations, aphasia, pyramidal syndrome, coma, cranial nerve palsy, dysautonomia, and central hypothyroidism. Notably, neurological disturbances were accompanied by laboratory evidence of CRS. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was undetectable in the cerebrospinal fluid (CSF). Hyperalbuminorrachia and increased levels of the astroglial protein S100B were suggestive of blood-brain barrier (BBB) dysfunction. Brain magnetic resonance imaging findings comprised evidence of acute leukoencephalitis (n = 3, one of whom had a hemorrhagic form), cytotoxic edema mimicking ischaemic stroke (n = 1), or normal results (n = 2). Treatment with corticosteroids and/or intravenous immunoglobulins was attempted, resulting in rapid recovery from neurological disturbances in two cases. SARS-CoV2 was undetectable in 88 of the 90 patients with COVID-19 who underwent Reverse Transcription-PCR testing of CSF. CONCLUSIONS: Patients with COVID-19 can develop neurological manifestations that share clinical, laboratory and imaging similarities with those of chimeric antigen receptor T-cell-related encephalopathy. The pathophysiological underpinnings appear to involve CRS, endothelial activation, BBB dysfunction, and immune-mediated mechanisms.

Estimating the incidence of fly myiases in Australian sheep flocks: development of a weather-driven regression model.

The blowfly, Lucilia cuprina Wiedemann (Diptera: Calliphoridae), is the primary myiasis (strike) fly of sheep in Australia. Most strike occurs in the anal-perineum area (crutch), but strike to the neck, shoulders, back and withers (body) is also important. Regression analysis was used to determine the extent to which the weekly incidence of flystrike can be explained by variations in fly abundance and/or recent changes in weather, pasture conditions or flock management. Strike and flock management data were collected by questionnaire surveys of 30-60 sheep properties in each of three major sheep-producing areas in southeastern Australia, namely, Gunning (southern New South Wales), Inverell (northern New South Wales) and Flinders Island (Bass Strait). After using simulation modelling to remove effects due to shearing, crutching and/or insecticide treatment, pasture growth index was found to be the most important explanatory variable affecting the incidence of all forms of myiasis. Others were average weekly air temperature, the amount and frequency of rainfall, relative humidity, dung quality index and a factor denoting seasonal effects. Together, these variables accounted for 48.4% of the variation in body strike, 56.8% of that in crutch strike and 51.9% of that in other forms of strike. Prediction was improved by the inclusion of additional lagged variables describing previous strike, fly abundance and fly activity. With these additions, the variation explained increased to 60.4% for body strike, 68.0% for crutch strike and 58.3% for other strikes.

Microsatellites reveal male recombination and neo-sex chromosome formation in Scaptodrosophila hibisci (Drosophilidae).

In drosophilid flies, male recombination and neo-sex chromosome formation are rare. Following the genotyping of full-sib families with 20 microsatellite markers and subsequent cytological work, we found evidence of both male recombination and neo-sex chromosome formation in Scaptodrosophila hibisci. As far as we are aware, this is the first report of male recombination and neo-sex chromosome formation co-occurring in a drosophilid fly. Two autosomal loci, Sh29c and Sh90, showed aberrant segregation of male parental alleles. We describe how an autosomal fission followed by fusion of one of the autosomal fragments to the Y chromosome to create a Y1Y2X1X2/X1X1X2X2 sex determination system provides the most parsimonious explanation of the patterns we observe. Male recombination was observed in three families, including autosomal linkage groups and the Y1/X2 linkage group. In addition to the X1 linkage group, two autosomal linkage groups were identified.

Cytogenetic variation in natural populations of the Old World screwworm fly Chrysomya bezziana (Diptera: Calliphoridae).

The existence of sibling species in the Old World screwworm fly Chrysomya bezziana would raise serious problems in eradicating this pest if it entered Australia. Cytogenetic variation in C. bezziana was investigated by analyzing pupal trichogen polytene chromosomes. Natural populations of C. bezziana spanning its range from southern Africa to Papua New Guinea were examined as well as hybrids between a New Guinea laboratory strain and natural populations. No evidence of sibling species was found. All populations exhibited the same basic banding pattern as the standard sequence established from a Papua New Guinea strain. Extensive asynapsis of chromosome homologues was found in some hybrid crosses and was therefore measured in all populations and hybrids to detect systematic variation. Asynapsis levels in most hybrids could not be statistically distinguished from those present in the parent populations except for crosses between populations at the ends of the range. This result does not permit asynapsis levels to be used in establishing the origin of introduced flies by estimating their distance from known populations. One inversion polymorphism and six band polymorphisms spread over three chromosomes were analyzed. Populations in each sampled region had characteristic combinations of band polymorphisms. This may offer a diagnostic method for determining the origin of flies accidentally introduced to Australia.

Cytogenetic variation in natural populations of the Old World screwworm fly Chrysomya bezziana (Diptera: Calliphoridae).

The existence of sibling species in the Old World screwworm fly Chrysomya bezziana would raise serious problems in eradicating this pest if it entered Australia. Cytogenetic variation in C. bezziana was investigated by analyzing pupal trichogen polytene chromosomes. Natural populations of C. bezziana spanning its range from southern Africa to Papua New Guinea were examined as well as hybrids between a New Guinea laboratory strain and natural populations. No evidence of sibling species was found. All populations exhibited the same basic banding pattern as the standard sequence established from a Papua New Guinea strain. Extensive asynapsis of chromosome homologues was found in some hybrid crosses and was therefore measured in all populations and hybrids to detect systematic variation. Asynapsis levels in most hybrids could not be statistically distinguished from those present in the parent populations except for crosses between populations at the ends of the range. This result does not permit asynapsis levels to be used in establishing the origin of introduced flies by estimating their distance from known populations. One inversion polymorphism and six band polymorphisms spread over three chromosomes were analyzed. Populations in each sampled region had characteristic combinations of band polymorphisms. This may offer a diagnostic method for determining the origin of flies accidentally introduced to Australia.

Differential replication of heterochromatin in polytene chromosomes of the Old World screw-worm fly, Chrysomya bezziana (Diptera: Calliphoridae), analysed by fluorescence staining.

The distribution and replication of heterochromatin in polytene trichogen chromosomes of the Old World screw-worm fly, Chrysomya bezziana, were studied using fluorescent staining techniques. Quinacrine and distamycin-DAPI, which selectively stain AT-rich DNA, and chromomycin, specific for GC-rich sequences, were used. Bright quinacrine and DA-DAPI fluorescence was found in the sex chromosome body and in all autosomal centromere regions. Chromomycin (CMA) staining results in very little bright fluorescence of the sex chromosome body and autosomal centromeric regions, but many bright bands of varying morphology are distributed in autosomal arms. The expected negative CMA staining of quinacrine and DA-DAPI bright regions was not found. The lack of reciprocal staining patterns may result from changes in the higher order chromatin structure of polytene chromosomes, or intercalation of divergent heterochromatic sequences. Comparison of the different staining techniques in mitotic and polytene cells shows that heterochromatin is differentially under-replicated, so that the proportions of the distinct fluorescent-specific chromatin changes during polytenization. CMA staining within autosomal arms suggests that repeated sequences intercalated in euchromatin are co-replicated during polytenization. The numerous fluorescent markers described also provide further morphological features for use in comparative cytological analysis of C. bezziana.

Polytene chromosomes of the Old World screwworm fly (Chrysomya bezziana) and its evolutionary relationships with Lucilia cuprina and Cochliomyia hominivorax (Diptera: Calliphoridae).

Standard polytene chromosome maps for the Old World screwsworm fly, Chrysomya bezziana, are presented. Good quality polytene chromosomes obtainable from pupal trichogen cells allow detailed analysis of autosomal euchromatin. The sex chromosomes are represented by irregular heterochromatic structures resembling those described previously in trichogen polytene chromosomes of the Australian sheep blowfly, Lucilia cuprina. A high degree of homology with the banding pattern of L. cuprina polytene chromosomes allowed direct recognition of approximately 60% of the L. cuprina complement in the C. bezziana maps. A further 13% may be homologous. The extensive homology observed is discussed in relation to the rate of chromosome rearrangement and conservation of karyotype elements in the evolution of Calliphorid flies. The observed conservation in polytene banding patterns should facilitate construction of phylogenies over a number of generic groups.

Nucleolar fragmentation in polytene trichogen cells of Lucilla cuprina and Chrysomya bezziana (Diptera: Calliphoridae).

The location of genes coding for 18S and 28S ribosomal RNA in mitotic and polytene cells of Lucilia cuprina and Chrysomya bezziana was investigated using in situ hybridization of an 18 + 28S ribosomal gene probe and silver staining. In both species ribosomal genes were localized to secondary constriction regions in sex chromosome heterochromatin. In L. cuprina mitotic cells the probe hybridizes to a distal secondary constriction region in the short arms of the X and Y chromosomes. In C. bezziana mitotic chromosomes ribosomal genes were located in distal secondary constriction regions in the long arms of the X and Y chromosomes. In polytene trichogen cells of both species, hybridization results varied with the level of polyteny. Cells of low polyteny have a single hybridization site, but with greater polytenization, increasing numbers of extrachromosomal fragments strongly hybridize to the ribosomal gene probe. No hybridization occurs in structures representing the sex chromosomes or in the autosomes. These results indicate that fragmentation and dispersal of the nucleolus occurs during polytenization. Silver staining of both unsquashed and squashed polytene nuclei show identical behaviour of multiple, varied-sized nucleolar bodies, thus confirming the in situ hybridization results. Uridine incorporation studies in L. cuprina indicated that transcription occurs in extrachromosomal bodies similar to nucleolar fragments. Nucleolar fragmentation is more pronounced in L. cuprina males, particularly in those with the translocation T(Y;2)540. Chromosomally normal C. bezziana show nucleolar fragmentation levels similar to that in L. cuprina males. Ribosomal genes are disproportionately replicated in trichogen cells to a much greater extent than surrounding heterochromatin. Nucleolar fragmentation may be a gene amplification system, but it is not known to what degree, relative to diploid amounts, ribosomal genes replicate in trichogen cells.

Characterization and chromosomal distribution of a tandemly repeated DNA sequence from the Australian sheep blowfly, Lucilia cuprina.

In the course of making a Lucilia cuprina genomic DNA library, a ladder of bands was seen in partial Sau3A digests. Complete digestion reduced this ladder to predominantly monomer units of approximately 190 bp. Nine independently isolated copies of this repeat were cloned and sequenced. Only two of these isolates are identical in sequence, the most divergent being 71% homologous. This satellite DNA occurs in all three wild-type strains tested, and, for the single case examined, in the embryonic, larval, pupal, and adult DNA. It represents approximately 3%-4% of the genome. Data obtained from in situ chromosome hybridizations indicate that this sequence is concentrated around the centromeric regions of the autosomes and over most of the sex chromosomes. Labelling is much stronger in mitotic compared with polytene chromosomes showing directly that this centromeric satellite DNA is grossly under-replicated during polytenization. This under-replication is even more pronounced on the sex chromosomes compared with the autosomes.

Cytological characterisation of heterochromatin in mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana (Diptera: Calliphoridae).

Mitotic and meiotic chromosomes of the Old World screwworm fly, Chrysomya bezziana, were studied using C-banding and quinacrine and counterstain-enhanced fluorescence techniques. The five autosomes in the karyotype are evenly graded in size, with somewhat variable arm ratios. Distinguishing all autosomes on these features alone can be difficult. C-banding produces small centromeric bands in the autosomes, whereas the much longer X and Y chromosomes have extensive dark C-band blocks with intermediate background staining. Most bright fluorescence occurs in the sex chromosomes, particularly the X chromosome, which has remarkable banding detail. Band resolution is greatly increased in mitotic metaphase cells from embryos. Quinacrine staining of mitotic chromosomes produces bright fluorescence at the centromere regions of chromosomes 2, 3, and 4, assisting in their identification. Meiotic chromosomes have distinctly reduced brightness and resolution of fluorescent bands and show marked chromatid asynapsis in the brighter regions of the sex chromosomes. Fluorochromes staining A.T-rich DNA (quinacrine and 4,6-diamidino-2-phenylindole (DAPI) produce bright staining in a large proportion of the sex chromosomes. By contrast chromomycin, which binds preferentially to G.C-rich DNA, stains a much smaller proportion of the sex chromosomes than expected from reciprocal staining. Together with the asynapsis data this indicates that much of the heterochromatin in the sex chromosomes has unusual structural properties.

Homozygous-viable pericentric inversions for genetic control of Lucilia Cuprina.

The isolation of homozygous-viable pericentric inversions for inclusion in field-female killing (FK) systems in Lucilia cuprina is described. From 7,236 irradiated chromosomes screened, 16 pericentric inversions were isolated. Four of these were viable as homozygotes. One of these, In (3LR) 14, possesses the properties required for inclusion in FK systems (tight linkage of one inversion break-point to the white-eye gene and substantial genetic exchange within the inversion in heterozygous females).

Differential sex chromosome replication and dosage compensation in polytene trichogen cells of Lucilia cuprina (Diptera: Calliphoridae).

Non banded sex chromosome elements have been identified in polytene trichogen cells of Lucilia cuprina using Y-autosome translocations, C-banding and Quinacrine fluorescence. The X chromosome is an irregular granular structure while the much smaller Y chromosome has both a dense darkly stained and a loosely organised segment. The X and Y chromosomes are under-replicated in polytene cells but comparison of C- and Q-banding characteristics of sex chromosomes in diploid and polytene tissues indicates that selective replication of non C-banding material occurs in both the sex chromosomes. Brightly fluorescing material in the Y chromosome is replicated to such an extent that it consists of half the polytene element, while the C-banding material, which makes up most of the diploid X chromosome, is virtually unreplicated. Differential replication also occurs in autosomes. In XXY males, and in males carrying a duplication of the X euchromatic region, a short uniquely banded polytene chromosome is formed. It is suggested that in males carrying two doses of X euchromatin a dosage compensation mechanism operates in which genes in one copy are silenced by forming a banded polytene chromosome.

C, Q and H-banding in the analysis of Y chromosome rearrangements in Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae).

In Lucilia cuprina C-banding produces procentric bands on all autosomes and deep staining over most of the X and Y chromosomes which conciderably facilitates the analysis of complex Y chromosome rearrangements. The Y chromosome is generally darkly C-banded throughout while in the X chromosome a pale staining segment is found in the distal portion of the long arm. Modulation of the banding reaction results in 'grey' areas in both X and Y. When C-banding is compared with allocycly it is clear that not all heteropycnotic regions in the sex chromosomes C-band to the same extent. Secondary constrictions in the short arms of both X and Y chromosomes are clearly revealed by C-banding, the X satellite being polymorphic for size.--Q-banding results in a brightly fluorescing band in the short arm of structurally normal Y chromosomes. This band loses its fluorescence in some translocations, probably through a position effect. Hoechst 33258 staining does not produce any brightly fluorescing bands.

Band and nucleolar polymorphisms in polytene chromosomes of Simulium ornatipes (Diptera, Simuliidae).

Polymorphisms for the presence or absence of supernumerary bands, for band size, nucleolar expression and the location of secondary nucleolar organisers found in the polytene chromosomes of Simulium ornatipes are described. In all the seven band polymorphisms analysed, six of which involve single bands and one a multiple band complex (IILH), phylogenetic evidence shows that there has been an addition of material. IILH consists of seven amplified C-bands, two supernumerary C-bands and a supernumerary segment involving two C-bands and an interband region. These bands are linked to the peracentric inversion IIL-3, five of the bands being located only within inverted segments. Comparison with mitotic chromosomes suggests that IILH heterochromatin is not under replicated in polytene chromosomes. Recombination between IILH components occurs at a very low level which is insufficient to disrupt the integrity of the polymorphism. It is concluded therefore that the complex evolved in a sequential series, the origin of IIL-3 being the first step. Single band polymorphisms, some of which are also linked to inversions, show similar heterochromatic properties to the IILH bands. A mechanism of selective DNA sequence amplification is proposed to explain increase in band size and the accompanying heterochromatinization. Most supernumerary bands may be amplifications of submicroscopic bands. Nucleolar organisers show heteromorphism for expression and rare secondary nucleoli are found on all chromosomes. It is argued that a multiplicity of sites for ribosomal genes are distributed in the genome and that selective sequence amplification, similar to that proposed above, can increase these sequences to a functional level at any of the sites. This would explain the lability of nucleolar sites in different blackfly species.

Polytene chromosomes in pupal and adult blackflies (Diptera: Simuliidae).

A number of pupal and adult tissues of eight Australian blackfly species representing three genera, Austrosimulium, Cnephia and Simulium, were examined for the presence of polytene chromosomes. Banded polytene chromosomes were found in malpighian tubules, hind gut, fat body, and ovary, but only those from the malpighian tubules of female adults and pupae were of good quality. A detailed comparison of polytene chromosomes from larval salivary glands and adult malpighian tubules was made in S. ornatipes and, to a limited extent, in S. melatum. The banding patterns of chromosomes from both tissues were found to be identical with minor differences in puffing patterns in S. ornatipes and chromocenter characteristics in S. melatum. A survey of the remaining six species shows five of them to have malpighian chromosomes suitable for detailed cytological analysis. Simultaneous studies of larval, pupal and adult polytene chromosome systems offer a novel approach to the analysis of population problems in blackflies. The ability to recognise sibling species in adults also has potential practical significance in efforts to control vectors of onchocerciasis.