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Our zebrafish facility employs an aggressive feeding protocol using Tecniplast Tritone robotic feeders throughout the fish life-span ( Fig.1 A), allowing for rapid rearing of zebrafish as our standard protocol. The use of the Tritone feeders also allows for precise and simple manipulation of daily feeding programs. Parental fish homozygous for the melanoma-inducing p53/BRAF mutations were incrossed, and 50 embryos were placed per 10 cm petri dish in E3 buffer until 6 days postfertilization (dpf) ( Fig.1 A). Some parental fish carried the Tg(crestin:EGFP) transgene, but this was not analyzed for this study ( Kaufman et al., 2016 ). Each dish was then reared in a 3.5 liter tank with excess rotifers beginning at 6 dpf until 10-14 days of life. Developing juvenile zebrafish were then fed via Tritone robot increasing aliquots of GEMMA pellet (Skretting)±rotifer culture ( Fig.1 A; TableS1 ). Importantly, to minimize any ‘jackpot’ effects of single tanks, all juvenile zebrafish born on the same day were mixed during 4-5 weeks of life, and then randomly redistributed with 25 individuals per 3.5 liter tank. These tanks were then moved from the nursery to the main system during the sixth week of life and assigned a feeding label providing four (4X), two (2X), and one (1X) time daily feeding of the same weight (60 mg) of GEMMA pellet per feeding ( Fig.1 A).

Fig. 1.

Feeding amount alters melanoma tumor onset in zebrafish model. (A) Feeding paradigm used to rapidly rear melanoma-prone zebrafish. (B) Gross tumor onset reported as Kaplan–Meier curves for percent (%) tumor-free survival at the indicated age (dpf). Number of zebrafish at risk are reported at 50 day intervals based on the most proximal prior observation. Reported values are based on the Log-rank (Mantel-Cox) Test as determined by Prism software where curves were generated. NR, not reached. (C) Zebrafish length at the indicated times for various adult feeding regimens. SL, snout to caudal peduncle length, is reported in centimeters with mean±standard deviation. (D) Representative images of zebrafish at 155 dpf in each feeding group. Scale bar: 1 cm.

As previously described, visual inspection of p53/BRAF zebrafish for raised lesions identifies melanoma tumors reproducibly, and this approach has been used to establish the role of multiple factors in modifying melanomagenesis ( Ceol et al., 2011 ; Lian et al., 2012 ). Thus, each cohort of zebrafish was scored for melanoma onset every 2weeks beginning at 9-10 weeks of age. The total number of animals in each tank was kept constant by replacing any tumor-bearing zebrafish, which were removed upon identification of a tumor, with similarly aged Casper or Na zebrafish. As shown in Fig.1 B, the rate of detectable melanoma was significantly increased in three independent cohorts of p53/BRAF melanoma-prone zebrafish fed four times daily (4X, median onset 180, 162 and 187 days) as compared to those fed two (median onset 222, 230 and 223 days) or one time daily (median not reached in any cohort). These were highly significant differences comparing the four versus two times daily ( P <0.0001, P <0.0001 and P <0.0013) and four versus one time daily ( P <0.0001 in each cohort) schedules. There was also a trend of quicker onset in the two versus one daily groups, although not significant in all cohorts ( P =0.22, P =0.0458 and P =0.0676). As all cohorts were fed our standard diet in the nursery, we did not intensively investigate effects on sexual maturity. The appearance of the melanomas in all groups at the time of tumor identification was qualitatively similar, and in this study, we did not track the behavior of established tumors in the different feeding groups. We measured a snapshot of size [(i.e. snout to tail, SL, length ( Parichy et al., 2009 )] of zebrafish in Cohort 1 at 155 and 211 days of life and found stable differences in size between the 4X and 2X/1X daily feedings ( Fig.1 C,D).

Of patients presenting to hospital with CAP, up to 10% will require critical care admission.

Streptococcus pneumoniae continues as the most common infective pathogen.

Staphylococcus aureus, Legionella , and gram-negative pathogens are increasingly frequent causative pathogens.

Combined viral and bacterial infections may induce a more severe spectrum of disease.

Severe community-acquired pneumonia (CAP) remains a frequent reason for admission to hospital. It is the most common cause of septic shock requiring escalation to treatment within an intensive care unit (ICU). Despite earlier recognition and recent advances in supportive care, severe CAP is still associated with substantial morbidity and mortality, more often seen in the elderly and those with considerable comorbidities.


CAP is defined as an acute infection of the pulmonary parenchyma, with symptom onset in the community. Diagnosis can still be made within 48 h of hospital admission to meet criteria for a community-acquired infection. Severe CAP is defined as a pneumonia requiring supportive therapy within a critical care environment, that is associated with a higher mortality rate. Severe CAP is frequently a multisystem disease and patients will often present with multiple organ failure.

The annual incidence of CAP is 1.6–10.6 per 1000 adult population in Europe. 1 Between 1.2% and 10% of patients requiring hospital admission to treat CAP will require ICU admission. The incidence of CAP increases with age, and more than 90% of deaths related to severe pneumonia occur in patients over the age of 70. The 28 day mortality rate in patients admitted to critical care is ∼17%, which increases to 24.4% in those requiring invasive mechanical ventilation and 28.8% in those that develop septic shock. 1 Mortality rates in younger patients are more influenced by the severity of the infection rather than the presence of comorbidities. Even in the absence of comorbidities, severe CAP is associated with excess mortality over subsequent years among survivors independent of age.

CAP classically presents with a triad of infective signs (fever, leucocytosis), clinical signs and symptoms (sputum production, tachypnoea, cough, pleuritic chest pain), and a new or changed infiltrate as observed on radiography, for which there is no other explanation except infection. However, these clinical signs and symptoms may not be universally seen or present typically, particularly in the elderly or immunosuppressed. Diagnosis of CAP may be clouded or complicated by underlying disease states that affect cardiorespiratory function and by atypical or subacute presentations of infection.

Pneumonia develops when the defensive mechanisms within the lung become overwhelmed by a pathogen which has been either inhaled or aspirated. This is more likely to occur with more virulent pathogens and in patients with reduced host defences. Pathogens responsible for CAP are varied and wide-ranging in their capacity to cause severe disease and extra-pulmonary features (Table 1 ). The predominant pathogen throughout all age groups remains Streptococcus pneumoniae. Legionella , gram-negative bacilli, influenzae species, and Staphylococcus aureus are becoming increasingly common causes of severe CAP requiring critical care admission in comparison with CAP managed outside of critical care units. The frequency of other less prevalent causes of CAP such as Chlamydophilia psittaci , Coxiella burnetii , and Mycoplasma pneumoniae varies according to epidemiological setting and in part on the diagnostic techniques that are used. No causative organism is identified in up to 36% of cases of severe CAP.

…a significant association between cobalamin [b12] status and performance on tests measuring fluid intelligence, spatial ability and short-term memory” with formerly vegan kids scoring lower than omnivorous kids in each case.

The deficit in fluid intelligence is particularly troubling, the researchers said, because:

…it involves reasoning, the capacity to solve complex problems, abstract thinking ability and the ability to learn. Any defect in this area may have far-reaching consequences for individual functioning.

I recognize that there are many reasons why people choose to eat the way they do, and I respect people’s right to make their own choices. I also know that, like all parents, vegetarians and vegans want the best for their children. This is why it’s absolutely crucial for those that abstain from animal products to understand that there are no plant sources of B12 and that all vegans and most vegetarians should supplement with B12. This is especially important for vegetarian or vegan children or pregnant women, whose need for B12 is even greater than adults.

there are no plant sources of B12

One of the greatest tragedies of the B12 epidemic is that diagnosis and treatment is relatively easy and cheap – especially when compared to treatment of the diseases B12 deficiency can cause. A B12 test can be performed by any laboratory, and should be covered by insurance. If you don’t have insurance, you can order it yourself from a lab like DirectLabs.com for $60.

As always, adequate treatment depends on the underlying mechanism causing the problem. People with pernicious anemia or inflammatory gut disorders like Crohn’s disease are likely to have impaired absorption for their entire lives, and will likely require B12 injections indefinitely. This may also be true for those with severe B12 deficiency causing neurological symptoms.

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may be as effective as injections for those with B12 malabsorption problems. However, most B12 experts still recommend injections for people with pernicious anemia and advanced B12 deficiency involving neurological symptoms.

Cyanaocobalamin is the most frequently used form of B12 supplementation in the US. But recent evidence suggests that hydroxycobalamin (frequently used in Europe) is superior to cyanocobalamin, and methylcobalamin may be superior to both – especially for neurological disease.

Japanese studies indicate that methylcobalamin is even more effective in treating the neurological sequelae of B12 deficiency, and that it may be better absorbed because it bypasses several potential problems in the B12 absorption cycle. On top of that, methylcobalamin provides the body with methyl groups that play an role in various biological processes important to overall health.

If you suspect you have B12 deficiency, the first step is to get tested. You need an accurate baseline to work from. If you are B12 deficient, the next step is to identify the mechanism causing the deficiency. This is something you’ll probably need help with from a medical practitioner. Once the mechanism is identified, the appropriate form (injection, oral, sublingual or nasal) of supplementation, the dose and the length of treatment can be selected.

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