Age-related changes in Egr1 transcription and DNA methylation within the hippocampus.

Aged animals show functional alterations in hippocampal neurons that lead to deficits in synaptic plasticity and changes in cognitive function. Transcription of immediate-early genes (IEGs), including Egr1, is necessary for processes such as long-term potentiation and memory consolidation. Here, we show an age-related reduction in the transcription of Egr1 in the dentate gyrus following spatial behavior, whereas in the area CA1, Egr1 is reduced at rest, but its transcription can be effectively driven by spatial behavior to levels equivalent to those observed in adult animals. One mechanism possibly contributing to these aging-related changes is an age-associated, CpG site-specific change in methylation in DNA associated with the promoter region of the Egr1 gene. Our results add to a growing body of work demonstrating that complex transcriptional and epigenetic changes in the hippocampus significantly contribute to brain and cognitive aging. © 2016 Wiley Periodicals, Inc.

Fear conditioning is associated with synaptogenesis in the lateral amygdala.

Fear conditioning in the rat typically involves pairing a conditioned stimulus (tone) with an aversive unconditioned stimulus (foot shock) which elicits a freeze response. Although the circuitry that underlies this form of learning is well defined, potential synaptic changes associated with this form of learning have not been fully investigated. This experiment examined synaptic structural plasticity in the lateral amygdala which is critical for the acquisition of the conditioned fear response. Adult male rats were randomly allocated to either a paired, unpaired or tone only condition. One day after the initial fear conditioning session and 1 h after a probe trial confirmation of a conditioned fear response, the rats were perfused and the relevant tissue was embedded for electron microscopic analysis. Synaptic changes were quantified in the lateral amygdala using a stereological approach. The results showed a significant increase in the number of synapses in the conditioned animals compared to controls. This finding suggests that an increase in synaptic compliment in the amygdala may underlie the acquisition of the conditioned fear response.

Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons.

Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomatous polyps, the precursors to colorectal cancer. APC and ras gene mutations have been shown to be important early molecular events in the development of colorectal neoplasms. The objective of this study was to establish the nature and frequency of these two genetic alterations in ACF harvested from human colorectal resection specimens. One hundred and fifty-four ACF comprised of between 1 and 56 crypts were harvested from the grossly normal mucosa of colorectal resection specimens of 28 patients with varying pathological diagnoses. One hundred and twenty-five ACF from 20 colons were screened for the presence of K-ras codon 12 mutations with a polymerase chain reaction/restriction enzyme-based method. The APC gene mutation cluster region was screened in 65 ACF from 20 colons using a polymerase chain reaction/single strand conformation polymorphism technique. Putative mutations were confirmed by direct sequencing. K-ras codon 12 mutations were identified in 13% (16 of 125) of ACF. We also identified APC mutations in 4.6% (3 of 65) of ACF. The results of this study demonstrate that both APC and K-ras mutations occur in ACF. These observations support the role of the ACF as a colorectal cancer precursor and provide further insight into the early genetic changes which occur during colorectal tumorigenesis.

Fluoroscopy-guided reduction and fibular nail fixation to manage unstable ankle fractures in patients with diabetes: a retrospective cohort study.

AIMS: Patients with diabetes are at increased risk of wound complications after open reduction and internal fixation of unstable ankle fractures. A fibular nail avoids large surgical incisions and allows anatomical reduction of the mortise. PATIENTS AND METHODS: We retrospectively reviewed the results of fluoroscopy-guided reduction and percutaneous fibular nail fixation for unstable Weber type B or C fractures in 24 adult patients with type 1 or type 2 diabetes. The re-operation rate for wound dehiscence or other indications such as amputation, mortality and functional outcomes was determined. RESULTS: Two patients developed lateral side wound infection, one of whom underwent wound debridement. Three other patients required re-operation for removal of symptomatic hardware. No patient required a below-knee amputation. Six patients died during the study period for unrelated reasons. At a median follow-up of 12 months (7 to 38) the mean Short Form-36 Mental Component Score and Physical Component Score were 53.2 (95% confidence intervals (CI) 48.1 to 58.4) and 39.3 (95% CI 32.1 to 46.4), respectively. The mean Visual Analogue Score for pain was 3.1 (95% 1.4 to 4.9). The mean Ankle Osteoarthritis Scale total score was 32.9 (95% CI 16.0 to 49.7). CONCLUSION: Fluoroscopy-guided reduction and fibular nail fixation of unstable ankle fractures in patients with diabetes was associated with a low incidence of wound and overall complications, while providing effective surgical fixation. Cite this article: Bone Joint J 2016;98-B:1197-1201.

Interaction of oxidized chicken ovotransferrin with chicken embryo red blood cells.

Iron-saturated chicken ovotransferrin was chemically oxidized with NaIO4, converting 50% of its methionine residues to their sulfoxide derivatives while maintaining 95% of its iron-binding activity. The oxidized chicken ovotransferrin was able to deliver iron to the chicken embryo red blood cell for heme synthesis. From competition experiments, oxidized diferric chicken ovotransferrin was estimated to be approx. 65% as efficient as unmodified diferric chicken ovotransferrin at competing with diferric (55Fe2) chicken ovotransferrin for the iron-donating sites of the chicken embryo red blood cells. The presence of apo chicken ovotransferrin preparations (native or oxidized) in the incubation medium had little effect on the rate of iron incorporation into heme from diferric chicken ovotransferrin. The effect of modifying the periodate-susceptible methionine residues in chicken ovotransferrin was small but significant. These methionine residues do not appear critical for the interaction of chicken ovotransferrin with the chicken embryo red blood cell receptors, the incorporation of chicken ovotransferrin into the cell, or the release of iron from chicken ovotransferrin for heme synthesis.

Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems.

The optional Escherichia coli prr locus encodes two physically associated restriction systems: the type IC DNA restriction-modification enzyme EcoprrI and the tRNA(Lys)-specific anticodon nuclease, specified by the PrrC polypeptide. Anticodon nuclease is kept latent as a result of this interaction. The activation of anticodon nuclease, upon infection by phage T4, may cause depletion of tRNA(Lys) and, consequently, abolition of T4 protein synthesis. However, this effect is counteracted by the repair of tRNA(Lys) in consecutive reactions catalysed by the phage enzymes polynucleotide kinase and RNA ligase. Stp, a short polypeptide encoded by phage T4, has been implicated with activation of the anticodon nuclease. Here we confirm this notion and also demonstrate a second function of Stp: inhibition of EcoprrI restriction. Both effects depend, in general, on the same residues within the N-proximal 18 residue region of Stp. We propose that Stp alters the conformation of EcoprrI and, consequently, of PrrC, allowing activation of the latent anticodon nuclease. Presumably, Stp evolved to offset a DNA restriction system of the host cell but was turned, eventually, against the phage as an activator of the appended tRNA restriction enzyme.

Multidisciplinary forensic approach--investigation of a neonaticide and alleged concomitant rape.

In the case presented, a young woman claimed to have spontaneously aborted a 30-weeks old fetus conceived after being raped by her cousin. The police investigation was centred on a probable neonaticide, mitigated by a possible rape. In Israel, the crime of rape carries a heavy punishment similar to that of first degree murder. Thus the implementation of the most recent scientific forensic techniques is of paramount importance in charging or clearing a suspected rapist. The skeletonized fetal remains were found four months after the reputed abortion. The alleged neonaticide was supported by the results of the forensic clinical and anthropological examinations, while the rape accusation was dismissed by the results of the molecular biology identification of the skeletonized fetal remains. The aim of this report is to encourage the forensic community to apply a multi-disciplinary approach, thus maximizing the decision making competence of the courts of law.

Isolation by polymerase chain reaction of a cDNA whose product partially complements the ultraviolet sensitivity of xeroderma pigmentosum group C cells.

A xeroderma pigmentosum (XP) cell line from complementation group C has been complemented to attain ultraviolet (UV) resistance and DNA repair proficiency, by transfection with a human expression cDNA library, followed by selection to UV resistance. We now show that the transfected cDNAs can be rescued from cellular DNA of a secondary transformant by its in vitro amplification using expression-vector-specific oligodeoxyribonucleotides as primers in a polymerase chain reaction. The amplified cDNAs were cloned into a mammalian expression vector. Their transfection into XP cells identified a single cDNA which specifically complemented the UV sensitivity of a group-C-derived cell line to the same partial UV-resistance levels exhibited by the transformant from which the cDNAs were rescued.

Complexity of sensory environment drives the expression of candidate-plasticity gene, nerve growth factor induced-A.

Exposure of animals to an enriched environment triggers widespread modifications in brain circuitry and function. While this paradigm leads to marked plasticity in animals chronically or acutely exposed to the enriched environment, the molecular mechanisms that enable or regulate such modifications require further characterization. To this end, we have investigated the expression profiles of both mRNA and protein products of a candidate-plasticity gene, nerve growth factor induced-A (NGFI-A), in the brains of rats exposed to increased environmental complexity. We found that NGFI-A mRNA is markedly up-regulated throughout the brains of animals exposed to the enriched environment, but not in the brains of either handled-only or undisturbed control groups. The most pronounced effects were observed in the somatosensory and visual cortices, in layers III and V, while more modest increases were observed in all other cortical layers, with the exception of layer I. A striking NGFI-A mRNA up-regulation was also observed in the striatum and hippocampal formation, notably in the CA1 subfield, of animals exposed to the enriched environment paradigm. Immunocytochemistry was also used to investigate the distribution of NGFI-A protein in response to the environmental enrichment protocol. A marked increase in the number of NGFI-A positive nuclei was identified in the enriched environment condition, as compared to undisturbed and handled-only controls, throughout the rat brain. While the greatest number of NGFI-A immunolabeled neurons was found in cortical layers III and V, up-regulation of NGFI-A protein was also detectable in layers II, IV and VI, in both the somatosensory and visual cortices. NGFI-A immunopositive neurons were also more numerous in the CA1 subfield of the hippocampal formation of animals exposed to the enriched environment, but remained at basal levels in both control groups. Our results implicate NGFI-A as one of the possible early genetic signals that ultimately lead to plastic changes in the CNS.

Upregulation of the immediate early gene arc in the brains of rats exposed to environmental enrichment: implications for molecular plasticity.

Exposure to an enriched environment, a procedure that induces plasticity in the cerebral cortex, is associated with pronounced morphological changes, including higher density of dendritic spines, enlargement of synaptic boutons, and other putative correlates of altered neurotransmission. Recently, it has been demonstrated that animals reared in an enriched environment setting for 3 weeks have less neuronal damage as a result of seizures and have decreased rates of spontaneous apoptosis. Even though clear morphological modifications are observed in the cerebral cortex of animals exposed to heightened environmental complexity, the molecular mechanisms that underlie such modifications are yet to be described. In the present work, we investigated the expression of the immediate early gene arc in the cortex of animals exposed to an enriched environment. Animals were exposed daily, for 1 h, to an enriched environment, for a total period of 3 weeks. Brains were processed for in-situ hybridization against arc mRNA. We found a marked upregulation of arc mRNA in the cerebral cortex of animals exposed to the enriched environment, when compared to undisturbed controls, an effect that was most pronounced in cortical layers III and V. Animals in an additional control group that were handled for 5 min daily, displayed intermediate levels of arc mRNA. Furthermore, arc expression was upregulated in the CA1, CA2 and CA3 hippocampal subfields and in the striatum, but to a lesser extent in the dentate gyrus of animals exposed to an enriched environment, as compared to the two control groups. Our results support the association between the upregulation of the immediate early gene arc and plasticity-associated anatomical changes in the cerebral cortex of the adult mammal.

Rapid kindling of the hippocampus protects against neural damage resulting from status epilepticus.

It has previously been reported that rats kindled via the standard kindling procedure do not exhibit the typical profile of neuropathology following status epilepticus. We wished to determine whether a 1-day rapid kindling procedure is also neuroprotective against cell damage resulting from prolonged seizure activity. We found that rats rapidly kindled from the dorsal hippocampus were more resistant to a kainic acid challenge 21-25 days after kindling than were unkindled control rats. Kindling prior to a kainic acid challenge also provided substantial protection against status epilepticus-induced damage in the CA3 region of the hippocampus and piriform cortex in most animals. Thus, despite the short kindling time period, rapid kindling is neuroprotective against status epilepticus-induced cell damage.

Light-induced zif268 expression is dependent on noradrenergic input in rat visual cortex.

In the present paper we investigated the role of the noradrenergic projection from the locus coeruleus on the expression of the immediate early gene zif268 in the visual cortex of rats exposed to ambient light stimulation. Local administrations of 6-hydroxydopamine (6-OHDA), a specific toxin directed against the catecholaminergic system, were performed in the locus coeruleus prior to visual stimulation. Animals were stimulated for 2 h by ambient light, after a 2-week dark adaptation period. Sham-operated controls displayed a massive increase in the number of zif268 positive cells after light stimulation. To the contrary, lesioned animals demonstrated a dramatic reduction in the number of zif268 positive nuclei across all cortical layers. A few scattered immunopositive nuclei were identified in cortical layer IV, however, this region also underwent a significant reduction in the number of zif268 immunopositive nuclei. Our results indicate that the noradrenergic system plays an important role in the expression of zif268 in the visual cortex of rats exposed to ambient light after dark isolation.

A new method for the automated detection of spontaneous otoacoustic emissions embedded in noisy data.

There is, as yet, no universally agreed upon method for the detection of spontaneous otoacoustic emissions (SOAEs). In this paper, we augment extant techniques by presenting a new automated approach. The new procedure reliably labels the spectral peaks as SOAEs, rejects noisy data such as that due to body movements or breathing, provides a subject-specific measure of the probability that a particular peak is erroneously identified as a SOAE, and can be accomplished on-line. Comparison of results with this new approach and with methods undertaken previously in this laboratory and elsewhere are made. The estimated prevalence of SOAEs for the new method is as large as that for any method previously proposed.

Prevalence of spontaneous otoacoustic emissions in adults revisited.

The potential influence of spectral analysis factors on estimates of the prevalence of spontaneous otoacoustic emissions (SOAE) was explored. The detection of a SOAE was influenced by two spectral factors: (1) the frequency resolution of the spectrum, and (2) the number of spectral averages. For 15 different combinations of these two factors, the estimate of the prevalence of SOAEs for females ranged from 64 to 81% and, for males, from 39 to 55%. Combining across all 15 combinations, 83% of the females and 62% of the males were judged to have SOAEs. These results provide a context for understanding the inconsistencies in previous estimates of the prevalence of SOAEs and underscore the need to employ many different spectral analyses because some SOAEs are only detected when the spectral analyses are suitably tailored.

Spontaneous otoacoustic emissions: measurement and data.

Sounds from the ear canal were measured and then analyzed off-line. A peak-picking algorithm located spectral maxima which might be designated as spontaneous otoacoustic emissions (SOAEs). The output from a 0.5-cc syringe, used to stimulate the volume of the ear canal, was also measured, analyzed and used to approximate the false-alarm rate of the measurement system. SOAE prevalence estimates depended on the false-alarm rate, just as the hit rate in a yes-no task does. With a false-alarm rate of zero, trends that have been found to be significant in the pooled results of other surveys were replicated: more SOAEs in the right ear, more SOAEs in females, and increased probability that the contralateral ear has an SOAE if the ipsilateral ear has an SOAE. In addition, many SOAEs failed to be detected in consecutive spectral analyses because they fluctuated in level.

Covariation of binaural, concurrently-measured spontaneous otoacoustic emissions.

Simultaneous recordings of binaural spontaneous otoacoustic emissions (SOAEs) were made for 2 female subjects. For SOAEs below about 3.6 kHz measured within a testing session, the frequencies of nearby monaural and binaural SOAEs tended to move in tandem, whereas widely separated SOAEs did not. Across many testing sessions spanning a menstrual cycle, all monaural and binaural SOAE frequencies shifted in tandem. Possible mechanisms consistent with these results are discussed.

Assay of sulfacetamide sodium ophthalmic solutions by high-pressure liquid chromatography.

A high-pressure liquid chromatographic method, using an absorption column and sulfabenzamide as the internal standard, is proposed for the determination of sulfacetamide sodium and its principal hydrolysis product, sulfanilamide, in eye drops. It affords an average recovery of 100.9% of added sodium sulfacetamide with a relative standard deviation of 1.9%.

Expression of the cDNA for the beta subunit of human casein kinase II confers partial UV resistance on xeroderma pigmentosum cells.

An immortalized xeroderma pigmentosum cell line belonging to the complementation group D (XP-D) was transfected with a normal human cDNA clone library constructed in a mammalian expression vector. Following UV-irradiation-selection, a transformant having a stable, partially UV-resistant phenotype was isolated. A transfected cDNA of partial length was rescued from the transformant's cellular DNA by in vitro amplification, using expression-vector specific oligonucleotides as primers in a polymerase chain reaction (PCR). Expression of this cDNA complemented the UV sensitivity of the XP-D cell line to the UV-resistance levels characteristic of the primary transformant. The nucleotide sequence of the cDNA was determined. The deduced protein identified the cDNA as encoding for the beta subunit of casein kinase II (CKII-beta). Similar to the effect exerted by the truncated CKII-beta cDNA, expression of a cDNA clone encompassing the complete translated region of CKII-beta leads to XP-D cells partially resistant to UV-irradiation. However, transfection of CKII-beta cDNA could also partially complement the UV-sensitivity of a xeroderma pigmentosum cell line belonging to group C (XP-C). Analysis by Southern, Northern and RNAase mismatch cleavage techniques did not reveal any functional defect in the CKII-beta gene of cell lines derived from either 7 XP-D or 10 XP-C families. We therefore consider it unlikely that either the XP-D or the XP-C DNA repair deficiency is associated with a defect in the beta subunit of casein kinase II. Nevertheless, our findings suggest the possibility that the cell's response to DNA damage is modulated by CKII-dependent protein phosphorylation.

An estimate of the prevalence of tinnitus caused by spontaneous otoacoustic emissions.

Prototypical experimental tests for linking spontaneous otoacoustic emissions with disruptive tinnitus have been described previously. Using similar experimental tests on 96 tinnitus sufferers, an estimate is made here of the prevalence of tinnitus caused by spontaneous otoacoustic emissions: the 95% confidence limits of this estimate are 1.11% and 9.05%.

Audible and annoying spontaneous otoacoustic emissions. A case study.

Four types of evidence indicate that spontaneous otoacoustic emissions (SOAEs) might be the basis of one patient's problematic tinnitus. First, when SOAEs were suppressed, the tinnitus was inaudible. Second, pitch matches to the lowest pitch of the tinnitus corresponded to the lowest frequency of the SOAE. Third, there was a more intense multicomponent SOAE in the right than in the left ear, and patient claimed that the tinnitus was louder in the right ear. Fourth, the patient's SOAEs were unstable and for this reason they might be audible. One practical consequences of this research is that patients with pathological tinnitus (which keeps them awake at night and interferes with concentration) should be tested for SOAEs. Because SOAEs are abolished by aspirin, it is possible that some unusual cases of problematic tinnitus could be easily treated.