Inaccuracies associated with the automated measurement of mean cell hemoglobin concentration in dehydrated cells

Because of discrepancies between electronically and manually measured values of mean cell hemoglobin concentration (MCHC) encountered in studies of pathologic red cells, we studied the effect of cell water content on MCHC measurements by both methods. A series of red cell samples with varying water contents (54%-164% normal) were prepared from normal cells using the antibiotic nystatin. MCHC was then measured, using the microhematocrit centrifuge and three different electronic cell counters in common laboratory use. For MCHC values above 36 g/dl as measured by the spun hematocrit method, all three electronic counters under estimmated the MCHC, with increasing error as the true MCHC increased. For MCHC values below 30 g/dl, the values from two conductivity based instruments agreed with those from the spun hematocrit method, whereas one instrument based on light scattering overestimated the MCHC. These results indicate that inaccuracies in the measured mean cell volume (MCV) of dehydrated or otherwise undeformable cells may lead to spurious values for MCHC when electronic cell counters are used.     

Degenerate target sites mediate rapid primed CRISPR adaptation

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.Bacteria and Archaea are regularly exposed to bacteriophages and other mobile genetic elements, such as plasmids. To control the competing effects of horizontal gene transfer, a spectrum of resistance strategies have evolved in prokaryotes (1). One of the most widespread and well-characterized are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated) systems, which provide bacterial “adaptive immunity” (1–8). Simply, CRISPR-Cas functions in three major steps. First, in a process termed “adaptation,” short sequences are derived from the invading element and incorporated into a CRISPR array (9). CRISPR arrays are composed of short repeats that are separated by the foreign-derived sequences, termed “spacers.” Second, CRISPRs are transcribed into a pre-CRISPR RNA (pre-crRNA), which is then processed into short crRNAs, which encompass portions of the repeats and most—or all—of the spacer. Finally, as part of a Cas ribonucleoprotein complex, the crRNAs guide a sequence-specific targeting of complementary nucleic acids (for recent reviews, see refs. 1–7).CRISPR-Cas systems are divided into three major types (I–III) and further categorized into subtypes (e.g., I-A to I-F) (10). The mechanisms of both crRNA generation and interference differ between the types and there are even significant differences between closely related subtypes. However, Cas1 and Cas2 are the only two Cas proteins completely conserved across all CRISPR-Cas systems and they are crucial for adaptation in Escherichia coli (10–12). The acquisition of new spacers is the most poorly understood stage in CRISPR-Cas immunity, mainly hindered by the paucity of robust laboratory assays to monitor this process (reviewed in ref. 9). Streptococcus thermophilus is highly proficient at spacer acquisition and provided much of the early insight into adaptation, showing that new spacers are typically acquired at one end of the CRISPR array from either phages (13–15) or plasmids (16). Recently, spacer acquisition has been detected in a variety of other systems (11, 12, 17–20). Adjacent to the expanding end of the array is the leader region, which harbors the promoter for pre-crRNA expression and sequences important for spacer acquisition (12, 21). Recent studies in E. coli in the type I-E system have shown that spacer acquisition can occur from phages and plasmids either when the Cas1 and Cas2 proteins are overexpressed or if the native cas genes are up-regulated, because of deletion of hns (11, 12, 20–22). The DNA targets (termed “protospacers”) of newly acquired spacers are consistently flanked by protospacer-adjacent motifs (PAMs), with the E. coli type I-E consensus 5′-protospacer-CTT-3′. PAMs were originally identified computationally (23) and were shown to play a role in interference in an early study (14). The importance of PAMs in the recognition and selection of precursor-spacers (prespacers) during adaptation was demonstrated unequivocally using assays that were independent of interference (12, 21). The simple overexpression of Cas1 and Cas2, in the absence of other cas genes, demonstrated these are the only Cas proteins essential for adaptation and are likely to recognize PAMs (12).Adaptation consists of two related stages, termed “naïve” and “primed” (9). Naïve adaptation occurs when a bacterium harboring a CRISPR-Cas system is infected by a new foreign element that it has not previously encountered. Although the acquisition of a new spacer can result in effective protection from the element, point mutations within the protospacer or PAM allow the element to escape CRISPR-Cas targeting (14, 24, 25). This aspect had been viewed as a weakness of CRISPR-Cas interference, but recent studies show that a positive feedback loop—called priming—occurs, which enables one or more new spacers to be acquired (11, 20, 22). Specifically, single mutations within either the PAM or the seed region of the protospacer, although inactive for interference, promote the rapid acquisition of new spacers from the same target (11). Priming is proposed to allow an effective response against viral or plasmid escapees through the incorporation of new spacers. Unlike naïve adaptation, priming is more complex, and in type I-E systems requires Cas1, Cas2, crRNA, the targeting complex termed Cascade [CRISPR-associated complex for antiviral defence, composed of Cse1, Cse2, Cas7, Cas5, and Cas6e (26–28)] and the Cas3 nuclease/helicase (11). Interestingly, the vast majority of spacers acquired through priming are derived from the same DNA strand as the original priming spacer (11, 20, 22). In addition, priming in E. coli was abolished by two mutations in the protospacer and PAM regions (11).In this study, we generated a mutagenic variant library of a protospacer and PAM region and used both individual high-throughput plasmid-loss assays and next-generation sequencing to determine the limits of both direct interference and indirect interference through priming. Our results demonstrate that direct interference tolerates mutations mostly at very specific positions in the protospacer, whereas priming tolerates extensive mutation of the PAM and protospacer regions. The results have wide evolutionary consequences for primed acquisition and could explain the retention of multiple “older” spacers in CRISPR arrays.     

Intracranial artery dissection

The aim of this narrative review is to evaluate the pathogenesis, clinical features, diagnosis, treatment and prognosis of intracranial artery dissection (IAD). IAD is a rare and often unrecognized cause of stroke or subarachnoid haemorrhage (SAH), especially in young adults. Two types of IAD can be identified: a subintimal or subadventitial dissection. It is suggested that a subintimal dissection results in luminal stenosis, thromboembolism and subsequently cerebral ischaemia, whilst a subadventitial IAD could result in the formation of a pseudo‐aneurysm and compression on brainstem or cranial nerves. Rupture of such a dissecting aneurysm causes SAH. The exact cause of IAD remains unknown but several factors are associated with its development. Diagnosis is based on clinical presentation and specific features seen on multimodal neuroimaging. The management of IAD depends on the clinical presentation. In the case of cerebral ischaemia, anticoagulants or antiplatelet agents are used, whilst in the case of SAH endovascular treatment is primarily advocated. Prognosis depends on clinical presentation. Presentation with SAH has a worse prognosis.     

Phytic Acid and Whole Grains for Health Controversy

Phytate (PA) serves as a phosphate storage molecule in cereals and other plant foods. In food and in the human body, PA has a high affinity to chelate Zn²⁺ and Fe²⁺, Mg²⁺, Ca²⁺, K⁺, Mn²⁺ and Cu²⁺. As a consequence, minerals chelated in PA are not bio-available, which is a concern for public health in conditions of poor food availability and low mineral intakes, ultimately leading to an impaired micronutrient status, growth, development and increased mortality. For low-income countries this has resulted in communications on how to reduce the content of PA in food, by appropriate at home food processing. However, claims that a reduction in PA in food by processing per definition leads to a measurable improvement in mineral status and that the consumption of grains rich in PA impairs mineral status requires nuance. Frequently observed decreases of PA and increases in soluble minerals in in vitro food digestion (increased bio-accessibility) are used to promote food benefits. However, these do not necessarily translate into an increased bioavailability and mineral status in vivo. In vitro essays have limitations, such as the absence of blood flow, hormonal responses, neural regulation, gut epithelium associated factors and the presence of microbiota, which mutually influence the in vivo effects and should be considered. In Western countries, increased consumption of whole grain foods is associated with improved health outcomes, which does not justify advice to refrain from grain-based foods because they contain PA. The present commentary aims to clarify these seemingly controversial aspects.     

小儿淋巴管瘤的MRI诊断

目的分析小儿淋巴管瘤的磁共振表现及其病理基础。方法对６例经手术病理证实的、年龄７天至７岁的小儿淋巴管瘤患者的磁共振表现进行回顾性分析。结果淋巴管瘤磁共振表现为Ｔ１ＷＩ上呈与肌肉相似或稍高的信号，Ｔ２ＷＩ上高于脂肪信号。５例瘤内可见低信号分隔，１例瘤内见血管流空影，３例见明显包膜，３例边界不清。２例病理诊断为海绵状淋巴管瘤，４例诊断为囊状淋巴管瘤。结论磁共振成像可较好地显示肿瘤的大小、形态及范围，从而指导手术治疗。     

Subcutaneous zygomycosis: report of 10 cases from two institutions in North India

Background Subcutaneous zygomycosis is an uncommon condition observed in tropics. Few series have been published, particularly from the northern regions of India. Objectives The aim of this study was to describe clinical, investigative and therapeutic details in subcutaneous zygomycosis observed in two teaching hospitals in Delhi. Patients and methods Ten patients seen over a period of 10 years (1999–2009) form the material for this report. Results There were four children and six adults. In four children, the presentation was a subcutaneous localized mass or gradually spreading plaque. In the others, it was observed over nasal region of face, spreading inward into mucosal sites and paranasal sinuses, and outward to the contiguous areas. Regional lymphadenopathy was present in two with facial lesions. Majority showed a granulomatous infiltrate with admixture of other cells, mainly eosinophils. Aseptate or poorly septate hyphae were observed in seven. In one patient in whom no hyphae were observed, there was dense perivascular inflammation. Organisms were cultured from four patients, Basidiobolus ranarum in two and Syncephalastrum racemosum in two. The main therapy used was a saturated solution of potassium iodide (KI). Four received only KI of which two attained cure after 3 months and 9 months respectively, and the other two showed signs of regression. In one boy subsidence was associated with reduced circumference of thigh. Ketoconazole or itraconazole was given with KI to hasten regression when response was slow or there were side‐effects to KI. Conclusion Awareness and early recognition will prevent disfigurement produced by advanced disease, misdiagnosis and unnecessary surgical intervention.     

Oral health-related quality of life before,during, and after orthodontic-orthognathic treatment: a systematic review and meta-analysis

Clinical Oral Investigations - The effect of combined orthodontic-orthognathic treatment was estimated, specifically the impact of pre-surgical orthodontic treatment, on oral health-related quality...