A COVID-19 monitoring process for healthcare workers utilizing occupational health.

BACKGROUND: Hospital-based occupational health (HBOH) is uniquely positioned to not only prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, but to care for healthcare workers (HCWs) sick with coronavirus disease 2019 (COVID-19). AIMS: The primary objective of this study is to describe a system where HBOH services were adapted to provide a monitoring programme whereby HCWs with SARS-CoV-2 received daily evaluations and treatment options in order to improve access to care, and to report the clinical outcomes and predictors of hospitalization in HCWs enrolled in the programme. A secondary objective is to compare clinical outcomes to data on national HCWs with COVID-19. METHODS: This retrospective cohort study used survey data collected on HCWs at a university health system with COVID-19 from 1 March 2020 through 1 December 2021. A firth regression model was used to examine the unadjusted and adjusted association between clinical factors and hospitalization. RESULTS: The study cohort included 4814 HCWs with COVID-19. Overall hospitalizations were 119 (2%), and there were six deaths (0.12%). Predictors of hospitalization include several co-morbidities and symptoms. A total of 1835 HCWs monitored before vaccine or monoclonal antibody availability were compared with data on U.S. HCWs in a similar time period. The monitored HCWs had a lower rate of co-morbidities (19% versus 44%, P < 0.001), a lower hospitalization rate (3% versus 8% P < 0.001) and case-fatality rate (0.11% versus 0.95% P < 0.001). CONCLUSIONS: This monitoring strategy for COVID-19 may be feasible for HBOH systems to implement and improve access to care, but more data are needed to determine if it improves outcomes.

Alcohol and drug use severity are independently associated with antiretroviral adherence in the current treatment era.

Substance use in people with HIV (PWH) negatively impacts antiretroviral therapy (ART) adherence. However, less is known about this in the current treatment era and the impact of specific substances or severity of substance use. We examined the associations of alcohol, marijuana, and illicit drug use (methamphetamine/crystal, cocaine/crack, illicit opioids/heroin) and their severity of use with adherence using multivariable linear regression in adult PWH in care between 2016 and 2020 at 8 sites across the US. PWH completed assessments of alcohol use severity (AUDIT-C), drug use severity (modified ASSIST), and ART adherence (visual analogue scale). Among 9400 PWH, 16% reported current hazardous alcohol use, 31% current marijuana use, and 15% current use of ≥1 illicit drugs. In multivariable analysis, current methamphetamine/crystal use, particularly common among men who had sex with men, was associated with 10.1% lower mean ART adherence (p < 0.001) and 2.6% lower adherence per 5-point higher severity of use (ASSIST score) (p < 0.001). Current and more severe use of alcohol, marijuana, and other illicit drugs were also associated with lower adherence in a dose-dependent manner. In the current HIV treatment era, individualized substance use treatment, especially for methamphetamine/crystal, and ART adherence should be prioritized.

Racial and ethnic disparities in COVID-19 disease incidence independent of comorbidities, among people with HIV in the US.

OBJECTIVES: To define the incidence of clinically-detected COVID-19 in people with HIV (PWH) in the US and evaluate how racial and ethnic disparities, comorbidities, and HIV-related factors contribute to risk of COVID-19. DESIGN: Observational study within the CFAR Network of Integrated Clinical Systems cohort in 7 cities during 2020. METHODS: We calculated cumulative incidence rates of COVID-19 diagnosis among PWH in routine care by key characteristics including race/ethnicity, current and lowest CD4 count, and geographic area. We evaluated risk factors for COVID-19 among PWH using relative risk regression models adjusted with disease risk scores. RESULTS: Among 16,056 PWH in care, of whom 44.5% were Black, 12.5% were Hispanic, with a median age of 52 years (IQR 40-59), 18% had a current CD4 count < 350, including 7% < 200; 95.5% were on antiretroviral therapy, and 85.6% were virologically suppressed. Overall in 2020, 649 PWH were diagnosed with COVID-19 for a rate of 4.94 cases per 100 person-years. The cumulative incidence of COVID-19 was 2.4-fold and 1.7-fold higher in Hispanic and Black PWH respectively, than non-Hispanic White PWH. In adjusted analyses, factors associated with COVID-19 included female sex, Hispanic or Black identity, lowest historical CD4 count <350 (proxy for CD4 nadir), current low CD4/CD8 ratio, diabetes, and obesity. CONCLUSIONS: Our results suggest that the presence of structural racial inequities above and beyond medical comorbidities increased the risk of COVID-19 among PWHPWH with immune exhaustion as evidenced by lowest historical CD4 or current low CD4:CD8 ratio had greater risk of COVID-19.

Anemia risk factors among people living with HIV across the United States in the current treatment era: a clinical cohort study.

BACKGROUND: Anemia is common among people living with HIV infection (PLWH) and is associated with adverse health outcomes. Information on risk factors for anemia incidence in the current antiretroviral therapy (ART) era is lacking. METHODS: Within a prospective clinical cohort of adult PLWH receiving care at eight sites across the United States between 1/2010-3/2018, Cox proportional hazards regression analyses were conducted among a) PLWH free of anemia at baseline and b) PLWH free of severe anemia at baseline to determine associations between time-updated patient characteristics and development of anemia (hemoglobin < 10 g/dL), or severe anemia (hemoglobin < 7.5 g/dL). Linear mixed effects models were used to examine relationships between patient characteristics and hemoglobin levels during follow-up. Hemoglobin levels were ascertained using laboratory data from routine clinical care. Potential risk factors included: age, sex, race/ethnicity, body mass index, smoking status, hazardous alcohol use, illicit drug use, hepatitis C virus (HCV) coinfection, estimated glomerular filtration rate (eGFR), CD4 cell count, viral load, ART use and time in care at CNICS site. RESULTS: This retrospective cohort study included 15,126 PLWH. During a median follow-up of 6.6 (interquartile range [IQR] 4.3-7.6) years, 1086 participants developed anemia and 465 participants developed severe anemia. Factors that were associated with incident anemia included: older age, female sex, black race, HCV coinfection, lower CD4 cell counts, VL ≥400 copies/ml and lower eGFR. CONCLUSION: Because anemia is a treatable condition associated with increased morbidity and mortality among PLWH, hemoglobin levels should be monitored routinely, especially among PLWH who have one or more risk factors for anemia.

Lessons learned from the design and implementation of myocardial infarction adjudication tailored for HIV clinical cohorts.

We developed, implemented, and evaluated a myocardial infarction (MI) adjudication protocol for cohort research of human immunodeficiency virus. Potential events were identified through the centralized Centers for AIDS Research Network of Integrated Clinical Systems data repository using MI diagnoses and/or cardiac enzyme laboratory results (1995-2012). Sites assembled de-identified packets, including physician notes and results from electrocardiograms, procedures, and laboratory tests. Information pertaining to the specific antiretroviral medications used was redacted for blinded review. Two experts reviewed each packet, and a third review was conducted if discrepancies occurred. Reviewers categorized probable/definite MIs as primary or secondary and identified secondary causes of MIs. The positive predictive value and sensitivity for each identification/ascertainment method were calculated. Of the 1,119 potential events that were adjudicated, 294 (26%) were definite/probable MIs. Almost as many secondary (48%) as primary (52%) MIs occurred, often as the result of sepsis or cocaine use. Of the patients with adjudicated definite/probable MIs, 78% had elevated troponin concentrations (positive predictive value = 57%, 95% confidence interval: 52, 62); however, only 44% had clinical diagnoses of MI (positive predictive value = 45%, 95% confidence interval: 39, 51). We found that central adjudication is crucial and that clinical diagnoses alone are insufficient for ascertainment of MI. Over half of the events ultimately determined to be MIs were not identified by clinical diagnoses. Adjudication protocols used in traditional cardiovascular disease cohorts facilitate cross-cohort comparisons but do not address issues such as identifying secondary MIs that may be common in persons with human immunodeficiency virus.

Sexual Assertiveness Scale (SAS) for women: development and validation.

Four studies were conducted to develop and validate the Sexual Assertiveness Scale (SAS), a measure of sexual assertiveness in women that consists of factors measuring initiation, refusal, and pregnancy-sexually transmitted disease prevention assertiveness. A total of 1,613 women from both university and community populations were studied. Confirmatory factor analyses demonstrated that the 3 factors remained stable across samples of university and community women. A structural model was tested in 2 samples, indicating that sexual experience, anticipated negative partner response, and self-efficacy are consistent predictors of sexual assertiveness. Sexual assertiveness was found to be somewhat related to relationship satisfaction, power, and length. The community sample was retested after 6 months and 1 year to establish test-retest reliability. The SAS provides a reliable instrument for assessing and understanding women's sexual assertiveness.

PIP: The construct of sexual assertiveness has potential for codifying the strategies women use to achieve sexual autonomy. The Sexual Assertiveness Scale (SAS) was developed to measure initiation of wanted sexual experience, refusal of unwanted sexual experience, and prevention of pregnancy and sexually transmitted diseases (STDs) with a regular partner. Four independent studies were conducted to establish the stability of the factor structure of the SAS, evaluate the set of predictors of sexual assertiveness, further assess construct validity in a population at high risk of STDs, and test reliability through two follow-ups. A total of 1613 US women from university and community populations were included in the studies. Confirmatory factor analyses indicated that Initiation, Refusal, and Pregnancy/STD Prevention remained stable across samples. Consistent predictors of sexual assertiveness were sexual experience, anticipated negative partner response, a history of sexual victimization as an adolescent or adult, and self-efficacy. Relationship satisfaction, power, and length were moderately related to sexual assertiveness. Finally, test-retest reliability was confirmed. Use of the SAS could facilitate the design of programs to help women to become more able to negotiate condom use, for example.

Triplex DNA in the nucleus: direct binding of triplex-specific antibodies and their effect on transcription, replication and cell growth.

Jel 318 and Jel 466 are triplex-specific monoclonal antibodies which previously have been shown to bind to cell nuclei and chromosomes by immunofluorescence. Their interaction was further characterized by two methods. First, isolated intact nuclei were encapsulated in agarose. Both antibodies showed significant binding to the nuclei which could be inhibited by adding competing triplex DNA but not by adding Escherichia coli DNA to which the antibodies do not bind. Both triplex-specific antibodies inhibited replication and transcription in the nuclei by about 20%. Secondly, the antibodies were introduced into synchronized myeloma cells by osmotic shock of pynocytic vesicles. Cell-cycle studies showed that the myeloma cells had an S phase of about 10 h and a doubling time of about 20 h. The cells were synchronized with thymidine and both cell growth and cell death were monitored. Introduction of the triplex-specific antibodies caused a marked decrease in cell growth without a significant increase in cell death. The effectiveness of the antibodies was improved by the addition of chloroquine diphosphate which inhibits degradation in the lysosomes. As a control, introduction of an antibody specific for a bacterial protein had little effect. In synchronized cells, inhibition of proliferation reached a maximum at 7 to 13 h after the release from the thymidine block. Thus, cells are most sensitive to the triplex-binding antibodies at the end of S phase and during G2. This result is consistent with the view that triplexes are involved in chromosome condensation/decondensation.

Characterization of a new monoclonal antibody to triplex DNA and immunofluorescent staining of mammalian chromosomes.

A monoclonal antibody, Jel 466, was prepared from mice immunized with poly[d(Tm5C)].poly[d(GA)]. The binding of Jel 466 to nucleic acids was characterized by solid phase radioimmunoassays and competition experiments. There was no binding to single-stranded DNAs or to duplexes which could not form triplexes. In addition, the antibody preferred the triplex form of poly[d(TC)].poly[d(GA)]; it bound weakly to the triplex derived from poly[d(G)].poly[d(C)], but there was no interaction with poly[d(T)].poly[d(A)].poly[d(T)]. This pattern of specificity is very different from that of Jel 318, a triplex-specific antibody that will bind to poly[d(T)].poly[d(A)].poly[d(T)]. The amino acid sequence of Jel 466 also showed very little homology with Jel 318, although both contain many positively charged amino acids. The immunofluorescent staining of mouse and human chromosomes with Jel 466 was studied. In all cases, there was a marked reciprocal relationship between the pattern of Jel 466 on the one hand and that of Hoechst 33258 and Jel 318 on the other. Jel 466 was negative for C-band and G-band but positive for R-band, whereas the opposite was found for Hoechst and Jel 318. Since C and G-bands are AT-rich and R-bands are GC-rich, these staining patterns match the sequence preferences of the two antibodies. Thus the base composition of triplex-forming DNA differs from domain to domain.

Plasmid dimerization mediated by triplex formation between polypyrimidine-polypurine repeats.

The ability of independent pyr.pur tracts to participate in triplex formation has been investigated in linear plasmids. The pyr.pur tract could be positioned at the ends of the plasmids or internally by a suitable choice of restriction enzyme. Dimer formation between plasmids was monitored by mobility shifts on agarose gels as well as by direct visualization in the electron microscope. Linear dimers and X and Y structures were observed. Control experiments showed that a pyr.pur tract was essential and was consistent with triplex formation in which the two pyrimidine strands were antiparallel. These structures were formed at pHs between 4 and 6, but once formed they remained stable up to pH 7. Spermine was required for formation of dimers at low ionic strength, but once formed the dimers remained stable in the absence of spermine. Additional linear plasmids were constructed with pyr.pur tracts at both ends; these formed structures at pH 4 which had mobilities identical to those of open circles. Triplex formation of this type may serve as a good model for loop formation in eukaryotic chromosomes.

Immunofluorescent localization of triplex DNA in polytene chromosomes of Chironomus and Drosophila.

Purine.pyrimidine (pur.pyr) DNA tracts are prevalent in eukaryotic genomes. They can adopt a triplex conformation in vitro under conditions that may exist in vivo, suggesting that triplex (H-) DNA may exist naturally in chromosomes. To explore this possibility and gain insight concerning potential functions, the distribution of triplex DNA was studied in fixed polytene chromosomes of Chironomus tentans and Drosophila melanogaster by indirect immunofluorescence microscopy using an anti-triplex DNA monoclonal antibody (Jel 318). Chromosomes stained with this antibody exhibited immunopositive regions corresponding to condensed chromatin bands; interbands were less immunofluorescent. These results imply that there is more triplex DNA in bands than in interbands. In Chironomus, nucleolar organizer regions and Balbiani rings were immunonegative, indicating that triplex DNA is not present in decondensed, transcriptionally active chromatin. A few specific bands in both Chironomus and Drosophila were intensely immunofluorescent. In Drosophila, one such region was 81F on chromosome 3R. Competition during staining with exogenously added sequences corresponding to a constituent 1.672 g/cm3 satellite DNA in region 81F failed to abolish the immunofluorescence, suggesting that the satellite DNA does not fortuitously react with Jel 318 and implying that unidentified pur.pyr sequences forming triplex DNA are also present at this location. Region 81F exhibits ectopic pairing with nonrelated chromosome regions that have also proven to be intensely immunopositive; this suggests that the formation of triplex DNA between common, shared pur.pyr sequences in these otherwise nonhomologous bands might account for the ectopic pairing phenomenon. Together with our previous results, these data are consistent with the hypothesis that triplex DNA may play a role in chromosome organization by participating in regional chromatin condensation.

Triplex DNA in plasmids and chromosomes.

Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.

Morphological and biochemical effects of endonucleases on isolated mammalian chromosomes in vitro.

Endonuclease digestion of isolated and unfixed mammalian metaphase chromosomes in vitro was examined as a means to study the higher-order regional organization of chromosomes related to banding patterns and the mechanisms of endonuclease-induced banding. Isolated mouse LM cell chromosomes, digested with the restriction enzymes AluI, HaeIII, EcoRI, BstNI, AvaII, or Sau96I, demonstrated reproducible G- and/or C-banding at the cytological level depending on the enzyme and digestion conditions. At the molecular level, specific DNA alterations were induced that correlated with the banding patterns produced. The results indicate that: (1) chromatin extraction is intimately involved in the mechanism of endonuclease-induced chromosome banding. (2) The extracted DNA fragments are variable in size, ranging from 200 bp to more than 4 kb in length. (3) For HaeIII, there appears to be variation in the rate of restriction site cleavage in G- and R-bands; HaeIII sites appear to be more rapidly cleaved in R-bands than in G-bands. (4) AluI and HaeIII ultimately produce banding patterns that reflect regional differences in the distribution of restriction sites along the chromosome. (5) BstNI restriction sites in the satellite DNA of constitutive heterochromatin are not cleaved intrachromosomally, probably reflecting an inaccessibility of the BstNI sites to enzyme due to the condensed nature of this chromatin or specific DNA-protein interactions. This implies that some enzymes may induce banding related to regional differences in the accessibility of restriction sites along the chromosome. (6) Several specific nonhistone protein differences were noted in the extracted and residual chromatin following an AluI digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunofluorescent staining of mammalian nuclei and chromosomes with a monoclonal antibody to triplex DNA.

Triplex DNA is an unusual conformation of DNA formed when two pyrimidine nucleotide strands share a common purine strand. A monoclonal antibody, demonstrated by numerous criteria to be specific for triplex DNA, was used to investigate the presence and distribution of this unique DNA configuration in nuclei and chromosomes of mouse LM cells and human lymphocytes. Indirect immunofluorescence microscopy revealed that constitutive heterochromatin in acetic-methanol fixed mouse nuclei was usually, but not always immunofluorescent, suggesting possible cell cycle related variations in the amount of triplex DNA or its accessibility in this condensed chromatin. In fixed mouse and human chromosomes, there was a positive correlation between immunofluorescent staining patterns, Hoechst 33258 banding, and G- and/or C-banding patterns. Unfixed, isolated mouse chromosomes also reacted positively with the antibody, particularly when they were gently decondensed by exposure to low ionic conditions at neutral pH. This result indicates that fixation is not mandatory for antibody staining, suggesting that some mammalian chromosomal DNA may be naturally organized in a triplex configuration. However, there is a possibility that fixation may facilitate the formation of additional triplex DNA complexes in potential sequences or expose previously inaccessible triplex DNA. The precise correspondence between the immunofluorescent patterns produced by anti-triplex DNA antibodies and G- and C-bands known to represent regions of chromatin condensation, suggests a potential role of triplex DNA in chromosome structure and regional chromatin condensation.

A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid. An antibody specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA.

Endonuclease banding of isolated mammalian metaphase chromosomes.

Evidence is presented that endonuclease digestion of isolated, unfixed chromosomes results in the production of banding patterns similar to those produced by digestion of fixed, air-dried chromosomes. Mouse L cell chromosomes were isolated under acidic or relatively neutral pH conditions, exposed in situ (as wet mounts on glass slides) or in vitro (in suspension) to micrococcal nuclease, Alu I or Eco RI, treated with a buffered salt solution, and stained with Giemsa. After any of these endonuclease treatments in situ, the centromeric regions of the chromosomes were intensely stained, characteristic of the C-banding observed in fixed chromosomes exposed to the same treatments. Although the fixed chromosomes were morphologically well-preserved after endonuclease digestion, the morphology of chromosomes digested in situ was variable, ranging from normal to swollen to highly distorted chromosomes. In the latter, the endonucleases induced dispersion of non-C-band chromatin; however, C-bands were still apparent as condensed, differentially-stained regions. Exposure of isolated chromosomes to Alu I in vitro also resulted in well-defined C-banding and led to the extraction of about 70% of the chromosomal DNA. From these results, the mechanism of endonuclease-induced C-banding appears to involve the dispersion and extraction of digested chromatin.

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility.

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility were examined in isolated mouse liver nuclei. To facilitate this study, a simple assay of chromatin structure was developed, based on the absorbances at 260 nm (A260) and 320 nm (A320) of nuclei in test solutions. By subtracting the A320 from the A260, a single "spectral index" was obtained which served as a useful, but not absolute, indicator of chromatin structure. Electron microscopy verified the validity of this approach. The results indicate that either 200 mM NaCl or 0.5 mM MgCl2 were capable of preserving the native 20 to 30 nm chromatin fiber structure. Below 200 mM NaCl, the native fiber progressively uncoiled to the 10 nm unit fiber. The presence of 0.5 mM MgCl2 inhibited this uncoiling. Only divalent cations stabilized condensed chromatin (heterochromatin) within the nucleus. Monovalent and divalent cations interacted with one another at critical concentrations and modified their individual effects on chromatin structure; e.g., 10 to 25 mM NaCl interfered with the action of 0.5 to 1.5 mM MgCl2, causing a complete loss of condensed chromatin. Maximum solubility of micrococcal nuclease-digested chromatin occurred at 10 mM NaCl, which treatment allowed the chromatin to unfold to the 10 nm fiber. However, ionic conditions that disrupted condensed chromatin but maintained the native chromatin fiber morphology still resulted in relatively high yields of soluble chromatin. Minimum solubility occurred under conditions which preserved the structure of condensed chromatin.

Fractionation of micrococcal nuclease-digested chromatin solubilized at physiologic ionic strength.

When mouse brain nuclei are optimally digested with micrococcal nuclease, most of the chromatin is soluble in a 180 mM salt/1 mM EDTA buffer [1]. At this ionic concentration, chromatin maintains its native structure [2]. In an attempt to selectively extract different fractions of chromatin from digested nuclei, we have examined the differential solubility of chromatin in the 180 mM salt buffer containing concentrations of MgCl2 ranging from 2 to 0 mM. The results suggest that digested chromatin may be fractionated into specific soluble chromatin fractions which correspond to nuclease-sensitive chromatin, bulk chromatin, and heterochromatin. These soluble fractions have a high molecular weight (up to 20 kbp), and contain a full complement of histones as well as a complex assortment of non-histone proteins. The residual insoluble fraction may be equivalent to a native, nuclear matrix-bound chromatin fraction.

Silver staining of histone-depleted metaphase chromosomes.

To investigate a possible relationship between the core-like structures seen in silver-stained chromosomes (prepared by standard cytogenetic methods) and the scaffolds observed in histone-depleted chromosomes, the ability of the scaffold to stain with silver has been examined. Isolated chromosomes were histone-depleted by washing in ammonium acetate or by spreading the chromosomes on an ammonium acetate hypophase. The residual chromosome structures were carbon-platinum shadowed or stained with silver, and then examined by electron microscopy. The results provide clear evidence that the scaffold structure has a high affinity for silver and is therefore similar in its silver-staining potential to the core structure in standard chromosomes. This suggests that the silver core in standard chromosomes may represent the scaffold visualized by histone depletion. The peripherally dispersed DNA radiating from the scaffold also proved to be silver-reactive, and additional experiments demonstrated that purified DNA is capable of binding silver. This result indicates that cytological silver staining is not simply a matter of staining protein, as has previously been thought, but may also involve the staining of chromosomal DNA. In the ammonium acetate-treated and carbon-platinum-shadowed preparations, the scaffold structure was highly variable in its morphology and appeared to be composed of undispersed or incompletely dehistonized chromatin fibers. The silver-stained scaffold reflected this variability. Taken together with other evidence, these findings lead to a questioning of the reality of chromosome core structures.